HK1069323B - Substances capable of potentiating laminin 5 productivity in epidermal cells and utilization thereof - Google Patents

Description

Substance capable of enhancing production of laminin 5 in epidermal cells and use thereof

Technical Field

The present invention relates to the field of cosmetics and skin science, and more particularly, to a composition for external application to the skin, which contains a substance capable of improving the ability of producing laminin 5 in epidermal cells, and also capable of cleaning the basal membrane of human skin and preventing skin aging. The present invention also relates to a method for preventing or improving skin aging using the above composition.

Background

As agents capable of enhancing the ability to produce laminin 5 in epidermal cells, soybean-derived preparations, lysolecithin, lysophospholipides, and the like have been known (JP-A-11-343226, JP-A-2000-226308). However, considering that there are various mechanisms involved in the maintenance of normal properties and functions of the skin, it is desirable to provide a substance capable of enhancing the production ability of laminin 5 in epidermal cells, which may fall within a different range from the above-mentioned agents or preparations, or may be derived from a different origin, if possible.

Disclosure of Invention

The object of the present invention is to provide a substance which may contain a compound in a different range from the aforementioned lysolecithin and which is capable of enhancing the ability to produce laminin 5, an anti-skin aging composition containing the substance, and a method for preventing or ameliorating skin aging at the site of topical administration when the composition is administered to the skin of a subject.

The present inventors have conducted investigations on various substances that can solve these technical problems and have promoted the ability to produce laminin 5. As a result, it has been found that hair-suppression action, inflammation-healing action, and anti-allergenicity action are generally known (refer to japanese patent application laid-open nos. 11-322548, 2-282332, and 2000-226332, respectively), but the inventors have also found that substances obtained by subjecting a specific extraction procedure to, in some cases, an extract from the roots of formonones containing isoflavoneglycosides, triterpenes, tannins, etc., an extract from trifolium containing coumarins, coumaric acids, etc., and an extract from adzuki beans containing saponins, etc., can enhance the ability of producing laminin 5, and can prevent and improve one of the causes of skin aging, i.e., can prevent and improve the decrease in "moisturization" and "tension" of the skin.

Further, the present inventors have found that a substance obtained by subjecting a plant extract selected from the group consisting of pueraria root extract, glycyrrhiza extract, blackberry lily extract, ジタノキ extract, イボツヅラフジ extract and fenugreek extract, which has been widely known as having an antioxidant effect and the like (Japanese patent application laid-open No. 6-24937), a cell activating effect and the like (Japanese patent application laid-open No. 60-23325), a cell activating effect and the like (Japanese patent application laid-open No. 7-138179), a collagen production promoting effect or a tyrosinase inhibiting effect (Japanese patent application laid-open No. 9-87164), a hyaluronic acid production promoting effect or a tyrosinase inhibiting effect (Japanese patent application laid-open No. 10-29923, Japanese patent application laid-open No. 9-30950), and a hyaluronic acid production promoting effect or a tyrosinase inhibiting effect (Japanese patent application laid-open No. 10-29924, Japanese patent application laid-open No. 9-30950), to a specific extraction operation, is applied to each, has the ability to enhance the production of laminin 5, and can prevent or improve the aging of skin, as well as the above-mentioned extract of the root of Ononia japonica. In addition, it was confirmed that the same activity was present in the non-plant-derived whey extract.

Further, since pueraria lobata extract (extract obtained from pueraria lobata by using 50% ethanol) has an action of promoting collagen synthesis of dermal fibroblasts, there is a report indicating that it is "a promising raw material having an action of improving skin wrinkles" (see h.mori et al, japan cosmetic technologist, 2000, osaka, drafts, p.25-27). The active ingredient for promoting the synthesis of collagen described in this draft is 4 ', 7-DHIF (4', 7-dihydroisoflavanone).

Furthermore, isoflavone glycoside (e.g., 8- β -D- グルコピラノシル -7-hydroxy-3 (3 ', 4' -dihydroxyphenyl) -4H-1-benzopyran-4-one) contained in the extract of Pueraria lobata has an effect of eliminating active enzyme in an evaluation system using enzyme, and thus it has been reported that it contributes to anti-skin aging (see Japanese patent laid-open No. 4-282305).

On the other hand, the present inventors have found that, to the best of the knowledge of the inventors, a plant extract never used as an active ingredient of an external preparation for skin, namely, a plant extract prepared from poppy ボコニア (a)Bocconia) Genus plant, Leguminosae プソフオカルプス (Psophocarpus) Genus plant, Leguminosae quassia (bitter wood)Cassia) Genus plant, Leguminosae エリスレア (Erythraea) Genus カンチヤラグア (CanchalaguaAnd the name learning:Erythraea chilensisorGentiana canchalagua) The extract of the plant selected from the group can also enhance the ability of producing laminin 5 in the epidermal cells, thereby preventing or improving skin aging.

Therefore, according to the present invention, there can be provided a composition for preventing or improving skin aging, which comprises an effective amount of a substance capable of enhancing the production ability of laminin 5 in epidermal cells, and an ingredient other than the substance, which is usually blended in a cosmetic or pharmaceutical skin external preparation.

The substance in the composition is one or more selected from the group consisting of plant extract or whey extract, except for extract of soybean seed.

Furthermore, the invention provides the use of such a substance for the preparation of a complex for preventing or ameliorating skin aging.

Also, a method for preventing or ameliorating skin aging is provided, which comprises topically administering an effective amount of the substance to the skin of a subject (e.g., a human) in whom prevention or amelioration of skin aging (e.g., inhibition of the formation of wrinkles or amelioration of wrinkles) is desired.

Drawings

Fig. 1 is a graph showing the effect of UVB of the (IV) シカクマメ extract described later on inhibiting the formation of skin wrinkles, by TEWL value comparison.

Fig. 2 is a graph showing the inhibitory effect by comparison of skin thickness.

Fig. 3 is a graph showing the effect of suppressing wrinkles by using the wrinkle score according to the visual judgment standard table of wrinkles.

Best mode for carrying out the invention

Laminin 5 is a major component of a structure composed of various glycoproteins and proteoglycans that are located at the boundary between the epidermis and dermis and are called epidermal basement membrane (Rousselle et al, j.cell biol.114, 567, 1991). It is known that laminin 5 causes epidermal blebbing due to abnormal gene deletionLaminin 5 is a protein essential for binding of the epidermis to the dermis (Aberdam et al, nat. gene., 6, 299, 1994). Also, it is known that laminin 5 promotes the formation of epidermal basement membrane (Tsunenaga et al, Matrix Biology 17, 603, 1998). The role of laminin 5 is not limited to theory. Specifically, laminin 5 contributes to the maintenance or repair of the normal basement membrane structure or its function, and can delay the progression of skin aging by repairing changes in the basement membrane structure caused by external stress such as ultraviolet rays and dryness, and internal stress such as mental stress in daily life. In other words, the enhancement of the above-mentioned laminin 5 production ability helps to maintain the normal structure of the human skin basement membrane, and helps to prevent or improve skin aging. The inventors of the present invention confirmed that the anti-aging agent of the present invention contributes to enhancement of the production of laminin 5 in epidermal cells, and to prevention or improvement of skin aging. The term "anti-aging" means preventing and improving the decrease in skin function associated with accumulation of structural changes of the basement membrane due to aging or photoaging, specifically preventing and improving wrinkles, sagging, and hardening of the skin. The "formononetin root" in the invention is prepared from the root of formononetinOnonis spinosaL.) (leguminosae: leguminosae) or a plant body called a formononetin root composed of roots and rhizomes of a plant having homology thereto. Therefore, the formononetin root extract is an extract extracted from such a plant body (crude drug is generally obtained) by using a lower alkanol such as 1, 3-butanediol and ethanol. However, the above-mentioned formononetin root extract may be an extract obtained by extraction from a plant other than the rhizome and root of formononetin root by other extraction methods, and may be included in the scope of the "formononetin root extract" in the present invention, as long as the object of the present invention is satisfied.

Further, the term "sweet clover" means sweet clover (Western エビラハギ) (e.g., Lu,Melilotus officinalisl, etc.) (leguminosae: leguminosae) or the same plant thereof, known as sweet clover. Therefore, the extract of Melilotus refers to a lower alkanol such as ethanol, propylene glycol, and 1, 3-butylene glycolLower alkylene glycol, etc., and extract obtained by extracting whole plant of Melilotus officinalis. Further, even an extract obtained by extraction from any plant body part other than the whole plant of trifolium, as long as the extract meets the object of the present invention, is included in the scope of "trifolium extract" described in the present invention.

The term "bean sprout" refers to soybean(s) (iii)Glycine maxMerill) or mung bean (Phaseolus radiatusL.) or red bean (Phaseolus angularisWight), etc. (leguminosae: leguminosae), or ブラツクマツペ, radish, alfalfa, buckwheat, etc. Therefore, the bean sprout extract means that it is extracted from these substances with various solvents.

The small bean extract is prepared from small beans in water or other solvent (A)Phaseolus angularisWight) or a homologous plant thereof. Preferred examples of such extracts include アズキ powder in the specification of the cosmetic category blend components.

Among the plant extracts of the present invention, those obtained by using, as an extraction solvent, a C1-C6 alkyl-C1-C6 alkyl ketone such as acetone or methyl ethyl ketone, or a C2-C6 alkanoic acid C1-C6 alkyl group such as ethyl acetate or butyl acetate are preferable.

The term "homologous plant" as used herein refers to a plant of a natural variety or close source of an exemplary species or strain, and the plant has the same active ingredient as the exemplary species or strain.

In addition, the stem of Pueraria lobata Ohwi (A) in the present inventionPueraria hirsutaMatsumura. orP.lobata.Ohwi orP. thunbergianaBenth.) extract, preferably from Pueraria lobata (Radix Puerariae) [ named (Kakkon) or Pueraria Radix)](the extract is obtained by cutting the storage root of Pueraria lobata Ohwi and drying, and can be prepared from commercially available crude drugs such as local products.) generally, it is preferable to use C1-C6 containing anhydrous methanol or ethanolExtracting the obtained extract with a secondary alkanol.

Further, the action of enhancing the ability of laminin 5 described in this specification is hardly or not recognized at all in the above-mentioned draft collection of Mori et al and isoflavone glycosides [ e.g., glycosides such as 4', 7-DHIF (or daidzein), daidzein (7-glucoside) and Puerarin (8-glucoside) ] obtained from pueraria lobata by an aqueous solvent such as hydrous ethanol described in japanese unexamined patent application publication No. 4-282305. However, the above-mentioned fraction extracted with methanol or ethanol or the like containing substantially no water according to the present invention has an effect of enhancing the ability of producing laminin 5, regardless of whether the above-mentioned isoflavone or isoflavone glycoside is contained. Here, "substantially free of water" means that even if water is contained, it contains only 10% or less of water at the maximum, preferably 5% or less, and more preferably 3% or less of water at the maximum. This means that the extracts obtained according to the present invention, particularly kudzu root itself and extracts derived from kudzu root, are significant as effective components showing an activity of enhancing the laminin 5 production ability, compared with the above glycosides and the like: contains lupeol (lupeol) and isopauerenol イソバウエレノ - ル (isobauerenol) with higher lipophilicity and the following structural formulas respectively.

Thus, according to Tai invention, Pueraria lobata (C.Y.)Pueraria hirsutaMatsumura) and homologous plants thereof (e.g.P. lobata Ohwi、P. thunnbergianaBenth), in particular from the root of kudzu (Kakkon or Pueraria Radix) itself or from fractions obtained from kudzu, for example from an extraction operation of kudzu with methanol or ethanol at room temperature, and dividing the extract thus obtained into fractionsThe fraction obtained from the organic solvent phase in a mixed solvent of water and an organic solvent substantially immiscible with water (e.g., ethyl acetate) or the fraction obtained by extraction from the above-mentioned partitioned aqueous phase with an organic solvent substantially immiscible with water (e.g., n-butanol), wherein the above-mentioned lupeol and/or materials rich in イソバウエレノ - ル are generally preferred for achieving the object of the present invention. It is needless to say that a purified product of lupeol and/or イソバウエレノ - ル may be used as the pueraria lobata-derived extract.

The Glycyrrhrizae radix extract is preferably Glycyrrhrizae radix extract foam, Glycyrrhrizae radix extract or Glycyrrhrizae radix flavonoid (oil soluble Glycyrrhrizae radix extract) of cosmetic type. Of course, from licorice root (Glycyrrhiza glabraL orGlycyrrhiza uralensisFisher) or homologous plants thereof, and are included in the range of the licorice extract of the present invention as long as the object of the present invention is satisfied. In addition to the cosmetic type blend component specifications, local licorice crude extract, local licorice extract, and the like can also be used.

The blackberry lily extract is prepared by using various solventsBelamcanda chinensisDC.) and the dried product of the rhizome of the homologous plant (crude drug name: blackberry lily).

ジタノキ extract, イボツヅラフジ extract and fenugreek extract are all plants native to Japan, ジタノキ (A), (B), (C) and (C) respectivelyAlstonia scholarisAlias name トバンノキ), イボツヅフラジ (TinosDora tuberculataBeumee), fenugreek (Trigonella foenum-graecum) ((R)Trigonella foenum-graecum) Or their homologous plant leaves, stems, branches, flowers, barks, seeds, fruits, rhizomes or whole plant, etc. by soaking or heating under reflux with extraction solvent, filtering, and concentrating. The plant extraction solvent used in the present invention may be any solvent generally used for extraction, and the organic solvent such as the above-mentioned alkanol (or ethanol), ester, or glycol may be used, or the above-mentioned organic solvent may be used as necessaryA mixed solution of an organic solvent and water. Typically ジタノキ extract is obtained by extracting the bark with ethanol, イボツヅラフジ extract is obtained by extracting the whole plant with 1, 3-butanediol, and fenugreek extract is obtained by extracting the seed with ethanol.

On the other hand, the whey extract is composed of components derived from whey obtained by treating mammalian whey with acid and ethanol to remove insoluble substances. Mammalian whey is preferably obtained by defatting milk and filtering with magnesium fluoride. The whey is preferably one or more selected from human, cow, goat, sheep and pig whey.

The extract is usually diluted with ethanol or the like and used as an active ingredient in the present invention, or the dried product is used as it is, or the dried product is redissolved in ethanol or the like and used as an active ingredient in the present invention.

Further, the raw material plants of the plant extract which can be used in the present invention and has not been used as a skin external preparation are as follows.

Papaveraceae ボコニアBocconia) Typical examples of the genus plant include パロ · アマリロ (Palo amarillilo, academic name:Bocconia arborea) (ii) a Leguminous プソフオカルプス (Psophocarpus) Typical examples of the genus plant include シカクマメ (academic name: psophocarpustetragonolobus) (ii) a Quassia of Leguminosae (A)Cassia) Typical examples of the genus plant include レタマ (Retama, academic name:Cassia spinecens) (ii) a Leguminous エリスレア (Erythraea) Genus カンチヤラグア (Canchalagua, academic name:Erythraea chilensisorGentiana canchalagua) Plants native to Peru may be mentioned. Among them, シカクマメ is also called wings マメ (ウイングドビ - ン), ミヤコグサ.

The plant extract is obtained by soaking or heating and refluxing the leaves, stems, branches, flowers, barks, seeds, fruits, rhizomes or whole plants of the above plants together with the extract solution, filtering, and concentrating. The extraction solvent used in the present invention may be any solvent generally used for extraction, and among them, alcohols such as methanol and ethanol are particularly preferable, and aqueous alcohols, and organic solvents such as acetone and ethyl acetate, or combinations thereof may be used alone, as the case may be. The extract thus obtained may be incorporated into the composition used in the present invention as it is or after dilution with ethanol or the like, or the dried extract may be incorporated into the composition used in the present invention as it is or after redissolution in ethanol or the like.

The extract as the active ingredient in the present invention has been exemplified above as the plant from which it originates, but even an extract of a closely-derived species or strain (homologous plant) derived from these plants is contained as the active ingredient in the present invention if it has a production ability of laminin 5 in epidermal cells equal to or greater than a specific extract described later.

The amount of the plant extract or whey extract added to the composition or skin preparation for external use of the present invention is not limited because the optimum amount varies depending on the form of the preparation, but may be limited to a relatively wide range in which the effect of the present invention is not affected by the addition. The amount of the compound is determined. If necessary, the activity of each extract is preferably evaluated in advance according to the method for evaluating the ability of laminin 5 to be produced, which will be described later. The term "if necessary" means that the content of the active ingredient in these extracts may not be constant depending on the place and time of picking the raw material. However, plant extracts commercially available in the form of crude drugs containing the above local formulations and cosmetic-type ingredients are generally relatively constant in the content of the active ingredient related to the present invention.

The composition or the external preparation for skin of the present invention may be in any form as long as it is suitable for external application to the skin or topical administration to the skin, and generally, ointment, cream, milky lotion, face toilet, パツク, bath agent, and the like are acceptable.

The composition or the external preparation for skin of the present invention may be formulated appropriately according to the above formulation, and if necessary, in addition to one or more of the above essential components, other components used in external preparations for skin such as cosmetics and drugs, for example, other laminin 5 production promoters, anti-aging agents, moisturizers, antioxidants, oily components, ultraviolet absorbers, surfactants, thickeners, preservatives, alcohols, PH adjusters, detergents, emulsifiers, powdery components, colorants, aqueous components, water, various skin nutrients, perfumes, and the like may be blended as necessary. The method of compounding these may be performed according to a method known per se.

Further, エデト acid ニナトリウム, エデト acid ナトリウム, sodium citrate, sodium polyphosphate, sodium metaphosphate, グルコン acid metal blocking agent, caffeine, tannin, verapamil, tranexamic acid derivative, various crude drugs, vitamin E acetate, グリチルリチン acid and its derivative or salt, グリチルレチン acid derivative, salicylic acid derivative, lysolecithin or lysophosphatidic acid, soybean preparation and other laminin 5 production promoter, glucose, fructose, mannose, sucrose, trehalose and other saccharide, arbutin, kojic acid and other whitening agent, ノニル acid ワレニルアミド, benzyl nicotinate, β -butoxyethyl nicotinate, capsaicin, gingerol, cantharis tincture, ichthammol, caffeine, tannic acid, α - ボルネオル, vitamin E nicotinate, vitamin E, Inositol nicotinate, cyclanolate, シンナリジン, tolazarin, acetylcholine, verapamil, tetrandrine, gamma-oryzanol, sulfur, 2, 7-dimethyl thianthrene, and other antiliporrhea agents, and for various purposes, Selagineol, zinc oxide, allantoin, Tulipa aromatica extract, Bupleurum extract, イブキジヤコウ extract, Belamcanda chinensis extract, Royal gambir extract, beech bud extract, casein hydrolysate, and rice extract may be addedHydrolysate, rice bran extract, peach kernel extract, クララ extract, thiotaurine, hypotaurine, marjoram's extract, silica-treated zinc, イチヤクソウ extract, xylitol, phycoerythrin and its hydrochloride, suberone, phellodendron extract, coptis extract, alkanna tinctoria root extract, peony extract, swertia extract, バチ extract, sage extract, loquat extract, carrot extract, aloe extract, mallow extract, iris extract, grape extract, coix extract, towel gourd extract, cucumber extract, saffron extract, ligusticum wallichii extract, ginger extract, forsythia suspensa extract, rosemary extract, garlic extract, capsicum extract, garden burnet extract, dried orange peel extract, orange peel, Vitamin A and vitamin B such as radix Angelicae sinensis, vitamin A acetate, etc₂Vitamin B butyrate₂Vitamin B such as flavin adenine nucleotide₂Vitamin B, vitamin C₆Vitamin B hydrochloride, ピリドキシンジオクタノエト, etc₂Vitamin C such as L-ascorbic acid, L-ascorbic acid dipalmitin ester, L-ascorbic acid-2-sodium sulfate, L-ascorbic acid phosphate, DL-alpha-vitamin E-L- アスコルビン, リン acid, ジエステルカリウム, calcium pantothenate, D-pantothenic acid panthenol, pantothenic acids such as パントテニルアルコル and パントテニルエチルエテル, and vitamin D₂Vitamin D₃Vitamins such as vitamin D, nicotinic acid, nicotinamide, nicotinic acid ベンジル, nicotinic acid, vitamin E such as alpha-tocopherol, vitamin E acetate, nicotinic acid DL-alpha-tocopherol, succinic acid DL-alpha-tocopherol, and vitamins such as vitamin P and vitamin H.

The composition or external preparation for skin of the present invention thus prepared can be administered to the skin 1 or more times per day and several days after each day, since the active ingredient contained therein does not substantially exert a bad influence on the human skin. In addition, a simple practical use test can be performed with the assistance of a volunteer or the like, and then an appropriate administration schedule is determined.

The present invention will be described below with reference to specific examples, but the present invention is not limited to the following examples.

(I)Preparation example of plant extract

Typical preparations generally available on the market as plant extracts are shown below.

(I-1) パロ & アマリロ extracts

50g of the woody portion of パロ & アマリロ was immersed in ethanol at room temperature for 1 week, and the extract was concentrated to obtain 7.2g of an ethanol extract.

(I-2) シカクマメ extract

50g of the seed fraction of シカクマメ was immersed in ethanol at room temperature for 1 week, and the extract was concentrated to obtain 4.6g of an ethanol extract.

(I-3) レタマ extract

レタマ g of flower and branch parts were immersed in ethanol at room temperature for 1 week, and the extract was concentrated to obtain 3.0g of ethanol extract.

(I-4) カンチヤラグア extract

50g of カンチヤラグア whole herbs were immersed in ethanol at room temperature for 1 week, and the extract was concentrated to obtain 5.2g of ethanol extract.

(I-5) preparation of an extracted or purified fraction derived from Pueraria lobata Ohwi

Extracting Radix Puerariae (Pueraria Radix product 1000g) with ethanol at room temperature to obtain extract

Extract (80g) (hereinafter also referred to as "kudzu root extract"). The obtained extract is distributed by water-ethyl acetate, and n-butanol is added into the water phase for solvent distribution again. The ethyl acetate phase and the n-butanol phase obtained were separated by evaporation of the solvent under reduced pressure to give an ethyl acetate fraction (18g) and an n-butanol fraction (38.5 g). Then, 1g of the ethyl acetate fraction was separated and purified by phase and difenoconazole phase シリカゲルカラム to obtain lupeol (24mg) and イソバウエレノ - ル (8 mg).

(II)Evaluation test for laminin 5-producing ability and results thereof

(1) Culture of epidermal keratinocytes

Epidermal keratinocytes were isolated from human pericarp and cultured in epidermal cell growth medium (KGM) with a low calcium concentration. The above medium was supplemented with bovine pituitary extract and EGF. After the cells were cultured in KGM to passage 4, adherent cells were suspended by treatment with trypsin EDTA, and aggregates of the cells were removed by filtration to obtain a uniform cell suspension. The cells were collected by centrifugation and resuspended in DMEM-F12 (2: 1) -0.1% BSA to 8X 10⁴And/ml. The cell suspension was added to 0.5ml of the same medium containing 0.5ml of a 2-fold concentration of the test agent. The culture was carried out at 37 ℃ for 24 hours using a 24-well plate. At the end of the culture, the culture supernatant was transferred to エツペンドルフチユ - ブ and stored at-20 ℃ until laminin 5 was measured. In addition, to make laminin 5 bound to cells and culture plastic soluble, each well was added with tris-hcl buffer (PH7.4) containing various surfactants and stored at-20 ℃ for 1 night. The next day, ultrasonication was performed, followed by refreezing. The next day, after redissolving, centrifugation was carried out at 10000rpm for 5 minutes, and the supernatant was transferred into a tube and stored at-20 ℃ until the time of measurement of laminin 5.

(2) Determination of laminin 5 by Honeycomb ELISA

Laminin 5 present in the culture supernatant, cell layer, was measured by the honeycomb ELISA method. The モノクロナル antibody, BM165, was bound to the laminin alpha 3 chain of laminin 5 on the solid layer of a 96-well ELISA plate. For the サンドイツチ laminin 5 assay, モノクロナル antibody against laminin β 3 chain, which was previously ビチオン -converted (b-6F12), was used as another antibody. In this method, only the ヘテロトリマ -mer (. alpha.3-. beta.3-. gamma.2) that had a role was measured, and the heterodimer (. beta.3. gamma.2) was not detected. The test reagent was added to each well to which a 3% ゼ チ ラ ン リン -phosphoric acid buffer solution containing b-6F12 had been previously added. The final dilution ratio in the wells of this reagent was culture solution 1/4, cell layer 1/10. The antigen-antibody reaction was carried out at 37 ℃ for 2 hours, and after washing the plate, a solution of avidin HRP (ホ - スラディシユパ - オキシダ - ゼ) was added, followed by reaction for 30 minutes to 1 hour. After washing, HRP in ABTS matrix was added and absorbance at 405nm was measured using ELISAO プレ - トリ - ダ -. A calibration curve is prepared within the range of 0 to 40 ng/ml.

The amount of laminin 5 produced is shown as a relative value with respect to the agent (control) to which no plant extract was added by calculating the sum of the amount of free in the medium and the amount remaining in the cell layer.

(3) Results

The results are shown in table 1 below. The test results of the fractions derived from the pueraria lobata extract are shown in the following tables 2 (the present invention) and 3 (comparative examples).

TABLE 1

TABLE 2Further processing or refining fraction of the extract of Pueraria lobata Ohwi of (I-5) above Activity (invention)

TABLE 3 Activity of isoflavones contained in Pueraria lobata (comparative)

*¹⁾: obtained from BIOMOL Research labs.

*²⁾8-beta-D- グルコピラノシル -7-hydroxy-3- (3, 4-dihydroxyphenethyl) -4H-1-benzopyran-4-one described in Japanese patent application laid-open No. 4-282305

(III)Skin aging symptom improvement effect confirmation test using formononetin root extract and as a result thereof

In this test, 22 healthy female volunteers aged 40-68 years were selected as test subjects, and the formation of wrinkles, fine wrinkles or a decrease in skin elasticity was significant during this period. We performed a follow-up test of the vanishing cream of example 1 described below on these 22 female volunteers (2 X.M., for 4 weeks). At the end of the test, the following skin characteristics were measured at constant temperature and constant humidity, compared with those before the application (when using the cosmetic product without the formononetin root extract).

Measurement items

The number and length of wrinkles were measured by extracting a copy of the external canthus with シリコ - ンレプリカ (Silflo, Flexico devilpments Co.).

The amount of keratin water was measured using コルネオメ - タ (Courage & Khazaka Co., Ltd.).

The viscoelastic properties of the skin were measured using キユ - トメ - タ (Courage & Khazaka Co.). The suction and release were repeated 3 times for 2 seconds and 1 second according to a usual method, and the elongation (Uf value) and the ratio of viscosity/elasticity (Uv/Ue value) of the skin at the 1 st and 3 rd times were compared with those before the use.

Furthermore, the adhesive tape was used to visually determine the extracted exfoliation of the horny layer, the skin texture uniformity, and the uniform exfoliation of the horny layer. Then, a questionnaire was made on the skin condition before and after the application.

Results (comparison before use with)

Analysis result of replica (detection; Paired Student t-test)

Wrinkle count-8% (p ═ 0.003)

Wrinkle Length-9% (p ═ 0.010)

The anti-wrinkle effect of the vanishing cream of example 1 was statistically considered significant.

Amount of keratin water (detection; Paired Student t-test)

+22％(p＝0.0001)

The moisturizing effect of the vanishing cream of example 1 was statistically considered significant.

Skin viscoelasticity (test; Paired Student t-test)

Uf (maximal amplitude, 1 st) -21% (p ═ 0.0063)

Uf (maximal amplitude, 3 rd time) -29% (p ═ 0.0007)

Uv/Ue-28％(p＝0.002)

It was confirmed that the use of the vanishing cream of example 1 significantly increased the elasticity of the skin.

Texture (detection; Paired Wilcoxon test)

Texture uniformity (Skin Network Sharpness) + 23%

(p＝0.0022)

Uniform exfoliation of Keratin (Skin Surface Aspect) + 33%

(p＝0.0001)

The skin texture improving effect of the vanishing cream of example 1 was considered to be significant.

Results of questionnaires conducted on volunteers

In the following items, the proportion of persons who felt to be effective

The softness of the skin is 91%

The reduction of wrinkles is 45%

Reduction of fine wrinkles by 77%

The texture improving effect is 77%

The soft feeling is improved by 82 percent

The elasticity is improved by 73 percent

Smoothness of skin 82%

The skin improvement effect is 95%

With the previously used products (extract of Mangifera indica root)

A combination of cosmetics) compared to which the more effective fruit is 90%

From the above, it is understood that the vanishing cream containing formononetin root shown in example 1 is an excellent anti-aging vanishing cream because of its anti-wrinkle, moisturizing and softness, increased elasticity and improved texture effects. In addition, since the safety was also confirmed, almost all volunteers felt it to be more effective than the previously used cosmetic with no formononetin root.

(IV)シカクマメInhibitory Effect of UVB of extract on wrinkle formation of skin Evaluation of

Test and results thereof

シカクマメ (see (I-2) of (I)), the inhibitory effect of UVB on the formation of wrinkles on the skin was evaluated. We divided 5 hairless mice into the following 3 groups, and the groups were divided so as to be as unbiased as possible in terms of body weight and skin condition.

Untreated group (non-irradiated UV, non-coated group)

UV irradiation + solvent (80% ethanol) coating groups

UV irradiation + 1% (w/v) シカクマメ ethanol extract coating group (prepared into 80% ethanol solution)

Method for changing the repeated UVB irradiation on the back of a mouse for wrinkle formation in a mouse (^*1) Is performed as part of (a). Irradiating the back of the mice (4 weeks old) in the UV irradiation group with UVB (light source: Toshiba FL-20SE fluorescent lamp, Toshiba electronics) 3 times per week for 10 weeks (according to conventional method)^*2、^*3) Then, 100. mu.l of the drug was applied 5 times per week. On the day of UVB irradiation, the test substance is coated with a chemical after UV irradiation in order to avoid the influence of UV absorption. In addition, the back was cleaned with ethanol to ensure that no drug remained on the skin surface and then UV-irradiated. The initial exposure was 36mJ/cm²At 2 weeks and then gradually increased to 216mJ/cm at 10 weeks²Once per time. The total irradiation dose was 4.6J/cm²。

10 weeks after the start of the UV continuous irradiation, the transdermal water transpiration amount (TEWL) and the skin thickness at the irradiated site were measured, and the above groups were compared by t-test (fig. 1 and 2). TEWL was measured using a Tewameter (product of Courage & Khazaka Co., Ltd.), and Peacock, Dail thichness Gauge (product of Kawasaki Co., Ltd.) was used for measuring the Thickness of the skin fat due to UV.

Then, the back of the mouse was photographed, followed by using a method of ビセット et al (^*4) In a state in which the group name of the animals was hidden, the animals were scored according to the judgment criteria of the wrinkle formation degree shown in the following table 4 (fig. 3). This work was performed individually by 3 panelists, and for the inter-group score, the U test of Mann-Whitney was performed.

TABLE 4

Visual judgment standard table for wrinkles

And (3) fractional: judgment criteria

0: wrinkles are not visible.

1: shallower, shorter, or fewer than 2 wrinkles.

2: shallow wrinkles can be seen.

3: deeper or longer than 2 wrinkles. Shallower, shorter, or fewer than the 4 wrinkles.

4: shallow wrinkles were visible on the entire skin surface.

5: deeper or longer than the wrinkles of 4. Shallower or shorter than the wrinkles of 6.

6: deep and long wrinkles can be seen.

7: wrinkles deeper and longer than 6 were added. Shallower or shorter than the wrinkles of 8.

8: deep and long wrinkles can be seen on the whole skin surface.

As is clear from fig. 1, the シカクマメ -extract coating group has an effect of promoting recovery against disturbance of skin barrier caused by light because the increase of the TEWL value is intentionally suppressed with respect to the solvent (80% ethanol) coating group (control).

In addition, as is clear from fig. 2, in contrast to the skin plumes formed by the UV control groups, intentional suppression was always performed in the シカクマメ extract coating groups.

Further, as is clear from fig. 3, the solvent (80% ethanol) coating group formed deep wrinkles. In contrast, the シカクマメ coating group of the present invention was statistically considered significant because wrinkle formation was significantly suppressed.

From the above description, it is understood that the シカクマメ extract has the effect of preventing or even suppressing skin disorders caused by ultraviolet rays.

Reference to the literature

Haratake A, Uchida Y, Schmuth M, Tanno O, Yasuda R, Epstein JH, Elias PM and Holleran WM: UVB-induced changes in permeability barrier function: effects of epidermal hyperproliferation and thymocyte-mediated responses, j. invent. dermatol.108: 769-775, 1997.

Naganumaa M, Yagi E and Fukuda M: induced generation of tryptic spots was delayed in UVB irradiated hairless mice, j.dermaltol.sci.25: 29-35, 2001.

Schwartz E: serial tissue transformation in the skin of uv-irradiated hairless mice, j. invest. dermatol.91: 158-161, 1988.

Bissett DL, Hannon DP and Orr TV: animal model of yang-aging skin: histological, physical and visible changes in the skin of uv-irradiated hairless mice, Photochemistry and Photobiology 46: 367-378, 1987.

Examples of the composition or external preparation for skin of the present invention

EXAMPLE 1 vanishing cream

(prescription)

A. 10.0% by weight

Cetyl octanoate 5.0

Triacontane 10.0

メドウフオ ム 3.0

テトラヒドロテトラメチルシクロテトラシロキサン 5.0

ジメチコンポリオ ル 3.0

クオタニウム 1 8ヘクトライト 2.0

Proper amount of perfume

エデト acid 0.1

Polyethylene glycol 60001.0

Glycerol 5.0

Dipropylene glycol 5.0

Tranexamic acid 0.5

Ascorbic acid magnesium phosphate 0.1

Sodium hyaluronate 0.01

L-phycocyanin hydrochloride 0.01

0.1 part of the extract of the root of Ononis

ミシマサイコ root extract 0.1

0.2 parts of methyl p-hydroxybenzoate

Ion exchange water surplus

The component B dissolved at room temperature was gradually added to the oil phase パツ in which the component a was uniformly dispersed, and the mixture was dispersed by a high-speed mixer.

EXAMPLE 2 wrinkle-preventing vanishing cream

(prescription)

Stearic acid 2.0% by weight

Stearyl alcohol 7.0

Aqueous lanolin 2.0

Triacontane 5.0

2-オクチルドデシルアルコ ル 6.0

ポリオキシエチレン(25モル)セチルアルコ ルエ テル 3.0

グ リ セ リン モ ノ ス テ ア リン acid エステル 2.0.0

Propylene glycol 5.0

0.05% ethanol extract of Melilotus officinalis

Ethanol extract of tulip 0.05

Sulfuric acid water solution ナトリウム 0.03.03

エチルパラベン 0.3

Proper amount of perfume

Ion exchange water surplus

(preparation method)

Propylene glycol was added to the ion-exchanged water and the temperature was maintained at 70 ℃ with heating (aqueous phase). Mixing other components, heating to dissolve, and keeping at 70 deg.C (oil phase). Adding oil phase into water phase, pre-emulsifying, uniformly emulsifying with high speed mixer, cooling to 30 deg.C while stirring.

EXAMPLE 3 vanishing cream

(prescription)

Solid paraffin 5.0 wt%

Beeswax 10.0

Vaseline 15.0

Liquid paraffin 41.0

グ リ セ リン モ ノ ス テ ア リン acid エステル 2.0.0

ポリオキシエチレン (20 モル) ソルビタン モ ノ ラ ウ リン acid エステル 2.0.0

Soap powder 0.1

Borax 0.2

モヤシアセトン extract 0.05

0.05 part of ethanol extract of bupleurum

Sulfuric acid water solution ナトリウム 0.03.03

エチルパラベン 0.3

Proper amount of perfume

Ion exchange water surplus

(preparation method)

Adding soap powder and borax into ion exchange water, heating to dissolve, and maintaining at 70 deg.C (water phase). Mixing other components, heating to dissolve, and keeping at 70 deg.C (oil phase). Mixing the mixture with the aqueous phase, and gradually adding the mixture to the aqueous phase for reaction. After the reaction is finished, the mixture is uniformly emulsified by a high-speed stirrer, and is cooled to 30 ℃ while being carefully stirred after emulsification.

EXAMPLE 4 vanishing cream

(prescription)

(A phase)

Stearic acid 10.0% by weight

Stearyl alcohol 4.0

Stearic acid butyl ester 8.0

ス テ ア リン acid モ ノ グ リ セ リン エステル 2.0.0

Vitamin E acetic acid 0.5

Vitamin A palmitate 0.1

マカデミアナツツ oil 1.0

Proper amount of preservative

Proper amount of perfume

(phase B)

Glycerol 4.0

1, 2-pentanediol 3.0

0.4 part of potassium hydroxide

Ascorbic acid magnesium phosphate 0.1

L-phycocyanin hydrochloride 0.01

エデト acid tris ナトリウム 0.05.05

Ethanol extract of red bean 0.1

Kudzu root 1, 3-butanediol 0.5

Ion exchange water surplus

(preparation method)

The oil phase of A and the water phase of B were heated to 70 ℃ respectively to completely dissolve them. Adding the phase A into the phase B, and emulsifying by using an emulsifying machine. The emulsion was cooled using a heat exchanger.

Example 5 emulsion

(prescription)

Stearic acid 2.5% by weight

Vaseline 5.0

Liquid paraffin 10.0

ポリオキシエチレン (10 モル) モノオレイン acid エステル 2.0.0

Polyethylene glycol 15003.0

Triethanolamine 1.0

カルボキシビニルポリマ 0.05

(trade name: カボポル 941, B.F. Goodrich Chemical company)

Glycyrrhiza acetic ether extract 0.01

Sulfuric acid water solution ナトリウム 0.01.01

エチルパラベン 0.3

Proper amount of perfume

Ion exchange water surplus

(preparation method)

Dissolved in a small amount of ion-exchanged water (phase a). Polyethylene glycol 1500 and triethanolamine were added to the remaining ion-exchanged water, and dissolved by heating to maintain 70 deg.C (aqueous phase). Mixing other components, heating to dissolve, and keeping at 70 deg.C (oil phase). Adding the oil phase into the water phase, pre-emulsifying, adding phase A, stirring with a high speed stirrer to emulsify, stirring and mixing, and cooling to 30 deg.C.

EXAMPLE 6 emulsion

(prescription)

(A phase)

Triacontane 5.0% by weight

オレイルオレト 3.0

Vaseline 2.0

ソルビタンセスキオレイン acid エステル 0.8.8

ポリオキシエチレンオレイルエ テル(20EO)1.2

Evening primrose oil 0.5

Proper amount of preservative

Proper amount of perfume

(phase B)

1, 3-butanediol 4.5

メリツサエタノル extractive solution 1.5

Ethanol 3.0

カルボキシビニルポリマ 0.2

0.1 part of potassium hydroxide

L-phycoproteinin L-asparaginate 0.01

Rhizoma Belamcandae ethanol extractive solution 1.5

ジタノキ 1 extraction of 1, 3-butanediol

Erythritol 0.5

Sodium hexametaphosphate 0.05

Ion exchange water surplus

(preparation method)

The oil phase of A and the water phase of B were heated to 70 ℃ respectively to completely melt. Adding phase A into phase B, and emulsifying with emulsifying machine. The emulsion was cooled using a heat exchanger.

Example 7 gel

(prescription)

95% ethanol 10.0% by weight

Dipropylene glycol 15.0

ポリオキシエチレン(50モル)オレイルアルコ ル

エ テル 2.0

カルボキシビニルポリマ (trade name: カボポル)

940，B.F.Goodrich Chemical company) 1.0

0.15 part of sodium hydroxide

L-phycocyanin 0.1

イボツヅラフジ 50% ethanol aqueous solution extract 7.0

Extract of fenugreek 90% ethanol in water 0.5

2-hydroxy-4- メトキシベンゾフェノン

スルホン acid ナトリウム 0.05.05

エチレンジアミンテトラアセテ ト·

3 ナトリウム.2 Water 0.05

0.2 parts of methyl p-hydroxybenzoate

Proper amount of perfume

Ion exchange water surplus

(preparation method)

カボポル 940 was uniformly dissolved in ion-exchanged water, and on the other hand, a 50% ethanol aqueous solution extract, a 90% ethanol aqueous solution extract of fenugreek, ポリオキシエチレン (50 モル) オレイルアルコルエテル was dissolved in 95% ethanol and added to the aqueous phase. Then, adding other components, and neutralizing and thickening with sodium hydroxide and L-phycocyanin.

EXAMPLE 8 cosmetic liquid

(prescription)

(A phase)

Ethanol (95%) 10.0% by weight

ポリオキシエチレン(20モル)

オクチルドデカノ ル 1.0

パントテニルエチルエテル 0.1

パロ & アマリロ メ タ ノ ル L extract 1.5

0.15 parts of methyl p-hydroxybenzoate

(phase B)

0.1 part of potassium hydroxide

(C phase)

Glycerol 5.0

Dipropylene glycol 10.0

Sulfuric acid water solution ナトリウム 0.03.03

カルボキシビニルポリマ 0.2

Purified water surplus

(preparation method)

Respectively dissolving phase A and phase C uniformly, and adding phase A into phase C to make it soluble. Then filling after adding B phase.

Example 9 toner

(prescription)

(A phase)

Ethanol 5.0% by weight

POE oleyl alcohol ether 2.0

Oleyl alcohol 0.1

2-エチルヘキシル P-ジメチル

アミノベンゾエト 0.18

Proper amount of perfume

(phase B)

1, 3-butanediol 9.5

Glycerol 2.0

Pyrrolidone carboxylic acid sodium 0.5

Nicotinic acid 0.3

シカクマメ 1, 3-butanediol extract 0.1

β-シクロデキストリン 1.0

Erythritol 0.05

Ion exchange water surplus

(preparation method)

Adding the alcohol phase of A into the water phase of B, and making it soluble to obtain cosmetic water.

Example 10 パツク

(prescription)

(A phase)

Dipropylene glycol 5.0% by weight

Polyethylene oxide (60 mol) hardened castor seed oil 5.0

(phase B)

レタマ 100% ethanol extract 0.01

カンチヤラグア acetic acid エチル extract 0.1

Olive oil 5.0

Vitamin E0.2

0.2 parts of methyl p-hydroxybenzoate

Fragrance 0.2

(C phase)

0.03

Polyvinyl alcohol

(degree of saponification: 90, degree of polymerization: 2,000) 13.0

Ethanol 7.0

Purified water surplus

(preparation method)

Respectively and uniformly dissolving phase A, phase B and phase C, and adding phase B into phase A to make it soluble. Then, the mixture is added to phase C and filled.

Example 11 solid Foundation

(prescription)

Talc 43.1% by weight

Argil 15.0

Sericite 10.0

Zinc oxide 7.0

Titanium dioxide 3.8

PMMA spherical powder 5.0

Yellow iron oxide 2.9

Red iron oxide 1.0

Black iron oxide 0.2

Triacontane 8.0

Isostearic acid 4.0

モノオレイン POE ソルビタン 3.0.0 acid

オクタン Tri イソセチル 2.0.2

0.5 part of the ethanol extract of the formononetin root

Proper amount of preservative

Proper amount of perfume

(preparation method)

Mixing pulvis Talci-black iron oxide powder with mixer, adding triacontane oily component, radix Ononiae Indicae ethanol extract, antiseptic, and perfume, mixing, filling into container, and molding.

EXAMPLE 12 emulsion type Foundation make-up (vanishing cream type)

(prescription)

(powder portion)

Titanium dioxide 10.3% by weight

Sericite 5.4

Argil 3.0

Yellow iron oxide 0.8

Iron oxide Red 0.3

Black iron oxide 0.2

(oil phase)

Decamethylcyclopentasiloxane 11.5

Liquid paraffin 4.5

ポリオキシエチレン Property ジメチルポリシロキサン 4.0.0

(aqueous phase)

Purified water 50.0

1, 3-butanediol 4.5

100% ethanol extract of radix Puerariae 1.5

ソルビタンセスキオレイン acid エステル 3.0.0

Proper amount of preservative

Proper amount of perfume

(preparation method)

Heating and stirring the water phase, adding the pulverized powder, and homogenizing. Then adding the oil phase after heating and mixing, homogenizing and mixing, adding the spice while stirring, and cooling to room temperature.

Industrial utilization of

As described above, the composition or the external preparation for skin of the present invention has an excellent effect of enhancing the laminin 5 production, has an excellent effect of reducing the skin function such as wrinkles, sagging, and hardening of the skin caused by the structural change of the basement membrane due to aging, photoaging, or the like, and can maintain the skin in an elastic or healthy state. Therefore, the present invention has utility in cosmetic or pharmaceutical manufacturing industries that provide these compositions.

Claims (4)

1. A composition for preventing or improving skin aging, which comprises an effective amount of a substance capable of enhancing the production ability of laminin 5 in epidermal cells, which is an extract of the plant winged bean (Psophocarpus tetragonolobus), said extract being obtained by using a compound selected from the group consisting of: the extraction solvent is selected from alcohols, aqueous alcohols, acetone, and ethyl acetate.

2. The composition according to claim 1, wherein the prevention or improvement of skin aging means inhibition of the formation of skin wrinkles or improvement of wrinkles.

3. A skin external preparation comprising an effective amount of an extract of plant winged bean and a component usually blended in a cosmetic or pharmaceutical preparation, wherein the extract is obtained by using a compound selected from the group consisting of: the extraction solvent is selected from alcohols, aqueous alcohols, acetone, and ethyl acetate.

4. Use of a substance capable of enhancing the production of laminin 5 in epidermal cells for the preparation of a composition for preventing or ameliorating skin aging, wherein the substance is an extract of the plant winged bean, said extract being obtained by using a substance selected from the group consisting of: the extraction solvent is selected from alcohols, aqueous alcohols, acetone, and ethyl acetate.