KR20200005055A - Composition for skin health comprising Glycine max (L.) Merrill variety SCEL-1 seed extract - Google Patents

Abstract

Provided are: a new variety bean SCEL-1 seed; a plant body and parts thereof; and an extract extracted from the same. A composition for skin health containing a bean variety SCEL-1 extract according to an aspect can be used for preventing or alleviating wrinkles and/or skin inflammation.

Description

Composition for skin health comprising Glycine max (L.) Merrill variety SCEL-1 seed extract}

It relates to a composition for use for skin health comprising a new breed soybean SCEL-1 seed extract.

Soybeans are a source of abundant plant protein and contain a variety of high functional substances, including unsaturated fatty acids, amino acids, isoflavones and phenolic acids, as well as proteins. For this reason, soy is used as a protein source to replace animal protein.

Cha Bae-Chun et al. (Journal of Pharmacognosy, 27 (3): 190-195 (1996)) is a wild type of soybean (Glycine soja Siebold et Zucc.) From the ethanol extract, the ethanol of soybean (Glycine max. Merr.) Unlike extracts, it has been found to contain (-)-epicatechin. In addition, it is disclosed that the ethanol extract of dol bean has anti-oxidative activity. However, chabaecheon et al. Do not disclose that soybeans contain (-)-epicatechin.

Therefore, there is a need for a composition comprising a new soybean variety, or extract obtained therefrom, that has superior antioxidant and / or other physiological functions as compared to conventionally known soybeans.

One aspect is the seed of the soybean ( Glycine max (L.) Merrill) variety SCEL-1, which has a higher procyanidin B2 content than cyanidin-3-O-glucoside, wherein a representative sample of the seed of the soybean variety is national. A composition for use for skin health comprising extract as an active ingredient an extract obtained by extracting seeds of soybean varieties deposited with KACC 98050P, deposited with the Academy of Agricultural Science, using water, alcohols of C1-6, or mixtures thereof. To provide.

Another aspect is a composition for use for skin health comprising cyanidin-3-O-glucoside, procyanidin B2, and epicatechin, or physiologically acceptable salts thereof, as an active ingredient, Glucosides, procyanidin B2, and epicatechin, or physiologically acceptable salts thereof, are based on weight by weight of cyanidin-3-O-glucoside or physiologically acceptable salts thereof, procyanidin B2 or physiologically acceptable salts thereof. And epicatechin or a physiologically acceptable salt thereof in a content ratio of 1: 2.0 to 2.1: 0.43 to 0.48.

Another aspect provides a method for preventing or improving skin wrinkle development, or preventing the occurrence of skin inflammation or improving skin inflammation, comprising administering a composition to the subject.

The first aspect comprises Cyanide-3-O-glucoside, procyanidin B2, and epicatechin, and a Glycine max (L.) Merrill variety that has a higher procyanidin B2 content than cyanidin-3-O-glucoside Seeds of soybean varieties in which the representative samples of the soybean varieties are deposited with the accession number KACC 98050P to the National Academy of Agricultural Science as water seeds of SCEL-1, alcohols of C1-6, or mixtures thereof. It provides a composition for use for skin health comprising the extract obtained by using the extract as an active ingredient.

In the seed, cyanidin-3-O-glucoside: procyanidin B2 is 1: 2.0 or more by weight, 1: 2.0 to 5.0, 1: 2.0 to 3.0, 1: 2.0 to 2.5, or 1: 2.0 to 2.1 Can be. The term "content" in the seed may be calculated based on the extract obtained by incubating the milled seed for 6 hours at 25 to 55 ℃ using 70 (v / v)% ethanol aqueous solution. As used herein, "content" is illustratively described, and it should be understood that there may be variations depending on the soil used to grow the plant from which the seed is obtained, climatic conditions, individual seeds, and the like.

In the seed, the procyanidin B2, and epicatechin total content may be at least 2 times, at least 2.5 times, for example, 2.6 times to 5.1 times higher by weight than the dark.

In the composition, the cyanidin-3-O-glucoside, procyanidin B2, and epicatechin content in the extract may be in an amount of 2.1 to 3.4% based on the total extract weight. Procyanidin B2, and the total amount of epicatechin in the extract may be 1.31 to 2.4% by weight of the total extract.

In the composition, cyanidin-3-O-glucoside, procyanidin B2, and epicatechin in the extract may be included in a content ratio of 1: 2.0 to 2.1: 0.43 to 0.48 by weight.

In the composition, the content of the extract, based on the total weight of the composition, 0.001% to 99.9%, 0.005% to 99.9%, 0.01% to 80.0%, 0.01% to 60.0%, 0.01% to 50.0%, 0.01% to 30.0%, 0.01% to 20.0%, 0.01% to 15.0%, 0.01% to 10.0%, 0.01% to 5.0%, 0.1% to 99.9%, 1.0% to 80.0%, 5.0% to 60.0%, 5.0% to 50.0% , 5.0% to 30.0%, 30.0% to 50.0%, 40.0% to 80.0%, 15.0% to 70.0%, or 50.0% to 90.0%.

When the seed is germinated to form a plant, it may have one of the properties listed in Table 1. Unless stated otherwise in the present specification, the characteristics of soybean varieties are necessary to explain the characteristics of the varieties of each crop according to Table 1 of the Seed Management Summary Article 2 of Article 30 of the Plant New Variety Protection Act and Article 33 of the Enforcement Decree of the Act and the Enforcement Rule of the same Act. "Soybean Glycine max (L.) Merrill", which defines the characteristics for the cultivation screening under Article 47, soybean Glycine max (L.) Merrill (National Species, Ministry of Agriculture, Food and Rural Affairs) , 2014: http.//www.seed.go.kr). Also, unless otherwise stated, quantitative traits among the characteristics of soybean varieties represent the mean value.

The composition does not include other extracts or active ingredients other than the above extracts, and may include the extract as the only extract or active ingredient.

A second aspect is a composition for use for skin health comprising cyanidin-3-O-glucoside, procyanidin B2, and epicatechin, or a physiologically acceptable salt thereof as an active ingredient, wherein cyanidin-3-O Glucoside or physiologically acceptable salts thereof, procyanidin B2 or physiologically acceptable salts thereof, and epicatechin or physiologically acceptable salts thereof in a content ratio of 1: 2.0 to 2.1: 0.43 to 0.48 by weight. It provides a phosphorus composition.

In the composition, the content of the cyanidin-3-O-glucoside, procyanidin B2, epicatechin or physiologically acceptable salt thereof is 0.001% to 99.9%, 0.005% to 99.9%, 0.01 based on the total weight of the composition. % To 80.0%, 0.01% to 60.0%, 0.01% to 50.0%, 0.01% to 30.0%, 0.01% to 20.0%, 0.01% to 15.0%, 0.01% to 10.0%, 0.01% to 5.0%, 0.1% to 99.9%, 1.0% to 80.0%, 5.0% to 60.0%, 5.0% to 50.0%, 5.0% to 30.0%, 30.0% to 50.0%, 40.0% to 80.0%, 15.0% to 70.0%, or 50.0% to 90.0 May be%.

The composition of the first and second aspects may be a food, cosmetic, or pharmaceutical composition.

In a second aspect, “physiologically acceptable salts thereof” includes “pharmaceutically acceptable salts”. The term "pharmaceutically acceptable" means that it can be used in animals, more specifically in humans, by avoiding significant toxic effects when used in conventional medical dosages. The term includes, for example, that can be approved or approved by the government or equivalent regulatory body, listed in the pharmacopoeia, or recognized as other general pharmacopoeia.

In a second aspect, the term "pharmaceutically acceptable salts" means salts according to one aspect of the invention that are pharmaceutically acceptable and have the desired pharmacological activity of the parent compound. The salt is formed from (1) an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, or the like; Or acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3- (4-hydroxybenzoyl) benzoic acid, shin Namsan, Mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4 -Methylbicyclo [2,2,2] -oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, Acid addition salts formed with organic acids such as hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid; Or (2) salts formed when the acidic protons present in the parent compound are substituted.

In the first and second aspects, an "active ingredient" means a component which alone can exhibit activity with a carrier which exhibits the desired activity or does not have activity on its own.

In the first and second aspects, “for use for skin health” means that the skin prevents wrinkles from developing or improves wrinkles by external stress, such as ultraviolet rays or oxidants, It indicates that the skin prevents or improves inflammation from external stress, such as ultraviolet light or oxidant, or internal stress.

In a composition for use for skin health, which is an aspect of the present specification, the composition includes preventing or treating skin wrinkles and / or skin inflammation.

The composition of claim 2, wherein the composition comprises an extract obtained by extracting seeds of soybean varieties deposited with the accession number KACC 98050P to the National Academy of Agricultural Science with water, alcohols of C1-6, or mixtures thereof as an active ingredient. It may be.

In one embodiment, the pharmaceutical composition may be a pharmaceutical composition effective for skin health, and specifically, may be a pharmaceutical composition effective for skin damage caused by ultraviolet rays or oxidative stress.

When the composition according to the present invention is applied to medicines, the composition can be formulated as an oral or parenteral dosage form in the form of solid, semi-solid or liquid by adding a commercially available inorganic or organic carrier as an active ingredient.

Examples of the preparation for oral administration include tablets, pills, granules, capsules, powders, fine granules, powders, emulsions, syrups, pellets and the like. In addition, preparations for parenteral administration include injections, drops, ointments, lotions, sprays, suspensions, emulsions, suppositories, and the like. In order to formulate the active ingredient of the present invention, it can be easily formulated according to the conventional method, and surfactants, excipients, coloring agents, spices, preservatives, stabilizers, buffers, suspensions, and other commonly used auxiliaries can be suitably used.

The pharmaceutical composition according to the present invention may be administered orally, parenteral, rectal, topical, transdermal, intravascular, intramuscular, intraperitoneal, subcutaneous.

In addition, the dosage of the active ingredient will depend on the age, sex, weight of the subject to be treated, the specific disease or pathology to be treated, the severity of the disease or pathology, the route of administration and the judgment of the prescriber. Dosage determination based on these factors is within the level of skill in the art. Typical dosages are 0.001 mg / day to 2000 mg / kg / day, more specifically 0.5 mg / kg / day to 1500 mg / kg / day.

In a composition for use for skin health, which is an aspect of the present specification, the composition includes a health food composition.

The composition may be processed into a drink, fermented milk, cheese, yogurt, juice, probiotic and health supplements including the same, and may be used in the form of various other food additives.

In one embodiment, the composition may contain other ingredients and the like that can give a synergistic effect to the main effect within a range that does not impair the main effect of the present invention. For example, it may further include additives such as perfumes, pigments, fungicides, antioxidants, preservatives, moisturizers, thickeners, inorganic salts, emulsifiers and synthetic polymer materials for improving the physical properties. In addition, it may further include auxiliary ingredients such as water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymer polysaccharides and seaweed extract. The above ingredients may be suitably selected and blended by those skilled in the art according to the dosage form or purpose of use, and the amount may be selected within a range that does not impair the object and effect of the present invention.

The formulations of the compositions according to the invention may be in various forms, such as solutions, emulsions, viscous mixtures, tablets, powders, and the like, which may be administered by various methods such as simple drinking, injection, spray or squeeze.

The composition comprises a cosmetic composition. The composition may be formulated in a parenteral dosage form. Parenteral dosage forms can be injections or external skin preparations. The external skin preparation may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal patch, drug containing bandage, lotion, or a combination thereof.

The external skin preparations are commonly used in external preparations for cosmetics and medicines, such as aqueous components, oily ingredients, powder components, alcohols, humectants, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, and fragrances. , Colorants, various skin nutrients, or combinations thereof, as appropriate.

The external preparation for the skin includes a metal blocker such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, belafamil, licorice extract, glablidine, and caline. Fruit hot water extract, various herbal medicines, drugs such as tocopherol acetate, glytilinic acid, tranexamic acid and derivatives thereof or salts thereof, vitamin C, magnesium ascorbic acid phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, Sugars, such as trehalose, can also be mix | blended suitably.

In a composition for use for skin health, which is an aspect of the present invention, the soybean SCEL-1 extract may include an extract of water, C1-C6 alcohol, or a mixture thereof of soybean SCEL-1, and specifically, soybean. Methanol, ethanol, propanol, butanol, pentanol, or hexanol extract of SCEL-1.

In the present specification, the soybean SCEL-1 extract may be a crude extract extracted using water, C1-C6 alcohol, or a mixture thereof as a solvent. The C1-C6 alcohol may be specifically methanol or ethanol. When the soybean is extracted with a solvent, the soybean may be extracted by adding about 2 to 15 times, about 3 to 15 times, about 5 to 15 times, or about 10 times the solvent. The extraction may be a heat extraction, cold extraction, reflux cooling extraction, or ultrasonic extraction, and the like, there is no limitation if the extraction is obvious to those skilled in the art. The extraction may be performed at room temperature, but may be performed under warm conditions for more efficient extraction. For example, it can be extracted at a temperature of about 40 to 100 ℃, about 40 to 80 ℃, about 40 to 60 ℃, about 50 ℃. Extraction time may be performed for about 2 to 8 hours, about 4 to 8 hours, about 5 to 7 hours, about 5.5 to 6.5 hours, about 6 hours. The temperature may vary depending on conditions such as extraction solvent and extraction temperature. The extraction may be extracted one or more times several times in order to obtain a larger amount of the active ingredient, for example, may be used to extract the combined extract of 1 to 5 times, 1 to 4 times, or three times consecutively.

In this specification, the soybean SCEL-1 extract may include a crude extract of soybean SCEL-1 as described above, and may be included as a soluble fraction of an organic solvent obtained by further extracting the crude extract with an organic solvent having a low polarity.

Hexane, methylene chloride, ethyl acetate, n-butanol, and the like may be used as the organic solvent. The extract extracted by the above method or a soluble fraction of the extract may be used as it is, but may be used in the form of a concentrate by filtration and concentrated, or may be used as a lyophilized form by lyophilization after concentration.

A third aspect provides a method for preventing or improving skin wrinkle development, or preventing the occurrence of skin inflammation or improving skin inflammation in a subject, comprising administering to the subject a composition of the first or second aspect. do.

The administration can be oral or non-oral administration. The method may be to prevent or improve the occurrence of skin wrinkles in the subject. The method may be to reduce the occurrence of or improve inflammation of the skin.

The method may be administering the composition to the skin of the individual. In this case, administration may be application or application to the skin. The method may be a method of makeup. The method may be to prevent or reduce the level of reactive oxygen species in the skin. The active oxygen species may be generated when ultraviolet light is irradiated to the skin.

The subject may be a mammal, for example a vertebrate, or a fish. The vertebrate may be a cow, a pig, a cat, a dog, or a sheep. The subject may be a mammal, not a human.

Compositions for use for skin health comprising the soybean variety SCEL-1 extract according to one aspect may be used to prevent or ameliorate wrinkles and / or skin inflammation.

A method for preventing or improving the development of skin wrinkles in an individual, or for preventing the development of skin inflammation or for improving skin inflammation in an individual, according to another aspect may be directed to preventing and improving the development of skin wrinkles in an individual, or to preventing the occurrence of skin inflammation. It can prevent or improve skin inflammation.

1 is a view showing the effect of the extract of the soybean selected to improve the wrinkles of the skin damaged by ultraviolet light in hairless mice.
Figure 2 shows the result of staining the tail tissue derived from hairless mice irradiated with ultraviolet light.
3 is a diagram showing the effect of soybean extract on the collagen density in the skin of the mouse and H & E stained with UV irradiation.

Hereinafter, one or more embodiments will be described in more detail with reference to Examples. However, these examples are intended to illustrate one or more embodiments by way of example, but the scope of the present invention is not limited to these examples.

Example  1: Fostering and Characterizing New Varieties

1. Breeding of varieties

Twenty particles of soybean seeds of genetically engineered soybean seed, collected from Dong-eup, Yongin-si, Gyeonggi-do, were flat. As a result of the cultivation, the soybean seeds were separated in flower shape and color, so that the traits were not fixed. For example, the flower color was white or pink and separated. Therefore, this bean seed is not recognized as a species because its trait is not fixed and there is no stability and uniformity.

Therefore, the present inventors grew the varieties by the net selection selection breeding method using the soybean seed. The selection was carried out from 2013 to 2017, and from 2013 to 2014 at the National Crop Experiment Station, Suwon, Rural Development Administration, and from 2015 to 2017. It was.

The net selection process from the collected soybean genetic resources is specifically, 20 soybean genetic resources are sown in early June in Rural Development Administration's test packaging every year from 2013 to 2016 and one individual for improvement of purity in late November. Were harvested as the sowing seeds for the following year. After four years of separation, the final fixed one was selected.

In order to confirm the uniformity, traits such as axillary color, flower color, hair color, pod color, leaf type, new type, flowering period and maturity were examined every year. Separation of embryonic axons and flower colors was observed in 2013 in the process of sequential separation of the collected gene sources, and no separation of the remaining traits was observed. Subsequents selected in 2013 were found to maintain uniformity in irradiation traits in the traits examined from 2014 to 2017. The distinction between the standard varieties (raw black) and the control varieties (Celadon No. 3) is broadly distinguished from the traits of new breed, number of days of flowering, embryonic axon, 100 grains and granular form as shown in Table 1. Table 1 shows the characteristics of the final net lineage. The data in Table 1 named the selected strain (variety) SCEL-1. Figure 4 is a photograph of the seed of the selected breed SCEL-1.

resource New breed type (Y / N) Days at the time of flowering Color (purple, white) Axle color (purple, green) Hair color
(Gal, gray) Longitudinal color Coloring 100 grains
(g) Standing Black (standard) Finite 66 Purple Purple Brown black black 11.6 rectangle Celadon No. 3 (Control) Finite 70 Purple Purple Brown black black 40.7 rectangle Selection breed (SCEL-1) infinite 58 White green Brown black black 8.8 An ellipse

2. Identification of varietal characteristics compared to control varieties

In order to compare the agricultural performance with the existing cultivars of SCEL-1, the performance of the cultivars was repeated in the yolk method 3 repetition using the celadon 3 as the reference cultivar in the 2017 Korea National Institute of Crop Science. Confirmed. The test plots were planted on June 8, 2017, planted after 4 lines of 60cm X 15cm, 2 copies per week, 4m long and planted. Won black bean (breed application publication number: 2010-341) is a species of melanoma and small-sized soybean, developed in 2009 by the National Institute of Food Science and Technology, which is similar to SCEL-1 selected as the most cultivated variety in farms. Cheongja No. 3 (breed application publication number: 2005-176), which is a cultivar which can be compared as a cultivar and used as a contrast cultivar, is a varieties of black soybeans, alleles, and soybeans developed in 2004 at the National Institute of Food and Nutrition. Used. The black bean and celadon 3 can be commercially purchased from the national species.

The traits examined for agricultural traits and performance tests were investigated mainly on quantitative component traits such as enteral, salvage, and basin. The quantitative component traits were examined for the length, number of sections, number of branches, narrow water, 100 grains, yield per week, quantity per gu, and quantity per 10a.

Tables 2 and 3 show the characteristics of the selected soybean varieties, standard varieties and contrast varieties.

resource
Length (cm)
Saving water (section)
Number of branches () The number of parties 1 side 2 narrow Three Black (standard) 56.6 ± 4.3 14.7 ± 1.7 6.7 ± 2.1 18.8 ± 2.3 201 ± 21 32 ± 15.2 Celadon No. 3 (Contrast) 71.9 ± 2.7 14.1 ± 0.3 7.3 ± 0.6 13.8 ± 4.9 81 ± 12 4.7 ± 0.8 Selection breed (SCEL-1) 82.9 ± 1.4 15.8 ± 0.5 6.4 ± 0.3 12.9 ± 2.3 133 ± 44 38.3 ± 8.6

resource
Total
Lip count
(G) in 100 tablets Per week
Quantity (g) Ward
Quantity (kg) 10a
Quantity (kg) Black (standard) 252 ± 31 512 ± 70 11.6 ± 0.2 32.9 ± 3.3 6.6 ± 0.7 587 ± 60 Celadon No. 3 (Contrast) 100 ± 12 191 ± 25 40.7 ± 3.1 26.7 ± 3.2 5.3 ± 0.6 477 ± 57 Selection breed (SCEL-1) 184 ± 54 397 ± 113 9.4 ± 0.4 23.1 ± 2.0 4.6 ± 0.4 413 ± 36

According to Table 2 and Table 3, the selected varieties were cut 26cm more in length and 1 more in water saving compared to the standard black. In addition, the number of branches was 6.4, similar to the standard varieties, and the ratio of 1-part, 2-part, and 3-parts was similar to that of the standard type. The total number of grains, the number of grains, the quantity per share, and the quantity per share were lower than the standard varieties of 184, 397 grains, 23.1g and 4.6g, respectively. Among 100 grains, it was 9.4g, which was smaller than the standard variety. Based on these results, the amount per 10a was calculated to be 413kg / 10a, which is 70% of the standard varieties, but 301% and 217% of the functional substances, procyanidin B2 and epicatechin, respectively. Specifically, the procyanidin B2 content was 149.4, 49.5, and 34.3 ug / 100mg in the selected species SCEL-1, far black, and celadon 3, respectively, and the epicatechin content was 46.9, 21.6, And 20.1 ug / 100mg. At this time, the procyanidin B2 content and the epicatechin content were confirmed by extraction according to the method of Section 1 of Example 2 below. At this time, the procyanidin B2 content and the epicatechin content were confirmed by extraction according to the method of Section 1 of Example 2 below.

Through this experiment, SCEL-1 has high uniformity, and is distinguished from existing varieties through comparison with standard varieties and control varieties, and in terms of agricultural traits and quantity and functional substance content in terms of agricultural traits and quantity. Selected varieties can be recognized as separate varieties.

The new varieties selected were named black small unidirectional soybeans as SCEL-1 and were deposited on March 15, 2018 with the accession number KACC 98050P. Figure 1 shows the growth of the bean varieties of the present application.

Example  2: extract obtained by extraction with alcohols of selected soybean varieties or mixtures thereof and uses thereof

In this example, extracts were prepared from seeds of the new soybean varieties SCEL-1 selected in Example 1, and their effects were confirmed.

1. Preparation of Extract

Seeds of the selected varieties were ground by a high speed grinder (Wonder Blender, 820 W, 30000 RPM, Sanplatec Corp) immediately before analysis. 1 g of the obtained soybean powder was mixed with 100 ml of an aqueous 70 (v / v)% ethanol solution in a glass tube, and stirred at 50 ° C. using a magnetic bar for 6 hours. The extract was then filtered with filter paper (Garde No. 131 Qualitative filter paper, Advantec).

Thereafter, the ethanol layer was dried for 1 hour with a nitrogen gas dryer (hurricane-Eagle, Cheongmin Tech) and then freeze-dried for 1 day to completely remove moisture. As a result, 0.19 g of soy extract (standard deviation SD: ± 0.015 ) In the experiment, the extract was dissolved at 30 mg / ml concentration in 50% v / v ethanol and used for HPLC analysis.

2. Confirmation of wrinkle improvement effect

Using the extract obtained in Section 1, the effect of improving wrinkles damaged by UV light was confirmed through experiments using EpiDrmFT ^™ , a 3D skin, and a hairless mouse model.

(1) Confirmation using artificial skin

Specifically, EpiDermFT ^™ , a three-dimensional (3D) skin manufactured by MatTek, was purchased and used in this experiment. The prepared 3D skin was filled with 900ul of the medium provided by the manufacturer on a 6-hole plate, and then placed on top of the plate, followed by incubation in an incubator at 37 ° C., 5% CO ₂ , and 100% humidity, and after 24 hours, UVB (40 mJ / cm ² ) Were examined and changed to a medium containing 10, 30, and 100 ug / ml of each test substance. After 72 hours of culture, the culture medium containing collagenase (MMP-1) and procollagen, which are secreted from artificial skin, was harvested and produced using a method according to the ELISA kit (AP biotech RPN2610). The amount of type I collagenase (MMP-1) was quantified. In addition, the amount of procollagen remaining without degradation using the same supernatant was quantified using the method according to the ELISA kit (Takara MK101). Each value is expressed as protein amount per ml. The results are shown in Table 4 and FIG.

Table 4 and Figure 1 is a view showing the effect of the soybean extract on the amount of MMP-1 and procollagen in UV-irradiated artificial skin.

sample Concentration (ug / ml) MMP-1 Biosynthesis (pg / ml) Remaining Procollagen Amount (pg / ml) No treatment group 3980 ± 250 19000 ± 320 UVB induction group
(40 mJ / cm ² ) 11800 ± 300 15000 ± 220 SCEL-1 Bean 10 9980 ± 220 17000 ± 130 30 8100 ± 230 19000 ± 260 100 7600 ± 180 22000 ± 379 Black Beans 10 11200 ± 350 16000 ± 400 30 10400 ± 320 17300 ± 480 100 8500 ± 350 18500 ± 500 Rat bean 10 10800 ± 340 16800 ± 496 30 10100 ± 380 18100 ± 550 100 8300 ± 220 19200 ± 460

As shown in Table 4 and FIG. 1, the treatment of SCEL-1 extract reduces the biosynthesis of matrix metalloprotease (MMP-1), a type I collagenase induced by UVB, and thus also prevents degradation of procollagen. I could see. Comparing the three soybeans, SCEL-1 showed a much better effect than the other black soybeans.

(2) Confirmation using animal model

By oral administration of the extract obtained in Section 1, the effect of improving skin wrinkles was confirmed through experiments using a hairless mouse model.

Eight week-old female hairless mice (Oriental Bio, SKH-1 species, average weight 26.2 g) were divided into seven groups in each group for the control and test groups, respectively. The experimental group was prepared by dissolving the lyophilized extract obtained in Section 1 in 2% ethanol, 1% Tween 80, and 0.8% carboxymethyl cellulose (CMC) aqueous solution. Mice were administered to 25 mg / kg / day, and 50 mg / kg / day. The control group was administered mice with 2% ethanol, 1% Tween 80, and 0.8% CMC aqueous solution on a volume basis. Administration was by direct administration into the stomach using an oral zoned. The soy extract was administered to mice in amounts of 25 and 50 mg / kg / day. Ultraviolet rays were irradiated after this administration.

A minimum erythema dose (MED) of 1 week of UV light in each group of mice for the control and test groups, respectively, with administration of the extract solution or 2% ethanol, 1% Tween 80, and 0.8% CMC aqueous solution. 3 weeks 2 weeks, 2MED three times a week for 2 weeks, 3MED twice a week for 2 weeks, 4MED twice a week for a total of 8 weeks. After oral administration of each sample for 8 weeks once daily, a skin wrinkle was produced by using silicone (SILFLO impression material, Flexico, Tokyo, Japan) to measure the wrinkle state (Visioline VL650, Courage + Khazaka, Koln, Germany) to observe the shape of wrinkles and to determine the value of wrinkle-related factors.

Mean form factor is the most accurate and reliable measurement method among wrinkle related factor values. Mean form factor is represented by analyzing the shadow of the wrinkles caused by the light source to the replica by depth and length.

Mean form factor = 1- (wrinkle depth / wrinkle length). At this time, this value is always between 0 and 1, and the closer to 1, the better the wrinkle improvement effect. The results are shown in FIG. 2 is a view showing the effect of the soybean extract of the present application on the level of wrinkle form and wrinkle related factors mean form factor in UV-irradiated artificial skin. As shown in FIG. 2, the group administered orally with SCEL-1 50mg / kg / day had a mean form factor value of 0.7, indicating that the effect of improving wrinkles was superior to the group ingesting other types of beans.

In addition, the skin tissue of the experiment-free hairless mice were subjected to H & E staining to observe the thickness of the skin tissue after formalin treatment, and the results are shown in Table 5 below. Masson Trichome staining was performed to measure the change in collagen, and the results are shown in Table 6 and FIG. 3. Figure 3 is a view showing the effect of the soybean extract on collagen density in the result of H & E staining of the mouse skin irradiated with UV and mouse skin.

sample Oral Dose (mg / kg / day) Epidermal thickness (um) No treatment group 15.5 ± 0.84 UVB (40 mJ / cm ² ) 55.2 ± 0.08 SCEL-1 Bean 25 46.5 ± 3.35 50 43.9 ± 1.19 General Black Bean 25 55.6 ± 0.98 50 55.7 ± 1.39 Rat bean
(Won-ju) 25 51.9 ± 2.17 50 55.0 ± 0.38

As shown in Table 5, the SCEL-1 treatment group had an effect of preventing the skin from becoming thick due to a significantly lower epidermal thickness of the skin during UV irradiation.

sample Oral Dose (mg / kg / day) Collagen amount (%) No treatment group 193.8 ± 5.6 UVB (40 mJ / cm ² ) 100 ± 7.1 SCEL-1 Bean 25 147.6 ± 11.5 50 176.1 ± 6.8 Black Beans 25 150.7 ± 5.1 50 161.0 ± 7.2 Rat bean 25 147.3 ± 4.7 50 159.2 ± 7.2

As shown in FIG. 3 and Table 6, the SCEL-1 treatment group was significantly superior to other soybeans in the activity of preventing collagen destruction and recovery of collagen upon UV irradiation.

From the above results, SCEL-1 extract showed an excellent effect of improving skin tissues and wrinkles damaged from ultraviolet rays.

3. Ingredient Analysis of Soybean Extract

Soybean extracts were subjected to mass spectrometry to analyze the active ingredients.

Specifically, component analysis of the soybean extract was performed using an Agilent 1260 HPLC system and a Bruker MicrOTOF-Q II mass spectrometer. The analytical column used Prevail C18 (250x4.6 mm, 5 um), where mobile phase solvent A was 95% water / 5% acetonitrile (0.1% formic acid) and mobile phase solvent B was 95% acetonitrile / 5% water ( 0.1% formic acid) was used. Solvent flow rate was set to 0.7 ml / min. The concentration gradient conditions of the solvent used for component separation are shown in Table 5.

Minutes A (%) B (%) Flow rate (ml / min) Pressure (bar) 0.00 95.00 5.00 0.700 1000.00 3.00 95.00 5.00 0.700 1000.00 23.00 50.00 50.00 0.700 1000.00 28.00 0.00 100.00 0.700 1000.00 33.00 0.00 100.00 0.700 1000.00 33.10 95.00 5.00 0.700 1000.00 40.00 95.00 5.00 0.700 1000.00

The temperature of the column was maintained at 35 ℃ and the sample was injected with 10 μL. The mass spectrometer has a mass range of 50 to 800 m / z in ESI (+) mode, 8 L / min for nebulization gas, 180 ° C source gas temperature, +4500 V capillary voltage, cone (cone) The components in the soybean extracts were analyzed using a voltage of 35 V.

As a result, epicatechin and procyanidin B2 of the selected SCEL-1 varieties were 0.32 to 0.46% and 1.0 to 2.0%, respectively, which were much higher than 0.14 to 0.15% and 0.33 to 0.35% of the control varieties.

The content ratio of cyanidin-3-O-glucoside, procyanidin B2, and epicatechin of SCEL-1 varieties selected on the basis of cyanidin-3-O-glucoside, a representative substance of anthocyanin antioxidants, is 1: 2.0 to 2.1: 0.43. To 0.48. On the other hand, in case of far black, 1: 0.43 to 0.70: 0.19 to 0.25.

Claims (13)

Of soybean ( Glycine max (L.) Merrill) varieties SCEL-1, including cyanidin-3-O-glucoside, procyanidin B2, and epicatechin, and having a higher procyanidin B2 content than cyanidin-3-O-glucoside Seeds of soybean varieties in which the representative samples of the soybean varieties deposited with the accession number KACC 98050P to the National Academy of Agricultural Science were extracted using water, alcohol of C1-6, or mixtures thereof as seeds. A composition for use for skin health comprising the obtained extract as an active ingredient. The composition of claim 1, wherein the extract comprises cyanidin-3-O-glucoside, procyanidin B2, and epicatechin in an amount of 2.1 to 3.4% by weight of the total extract. The composition of claim 2, wherein the extract comprises cyanidin-3-O-glucoside, procyanidin B2, and epicatechin in a content ratio of 1: 2.0 to 2.1: 0.43 to 0.48 by weight. The composition of claim 1, wherein the content of the extract is 0.005% to 99.9% with respect to the total weight of the composition. The composition of claim 1, wherein the skin health is prevention of wrinkles or improvement of wrinkles. The composition of claim 1, which is a food, cosmetic, or pharmaceutical. Cyanidin-3-O-glucoside, procyanidin B2, and epicatechin, or a physiologically acceptable salt thereof, as a composition for use for skin health, comprising: cyanidin-3-O-glucoside or The physiologically acceptable salts thereof, procyanidin B2 or physiologically acceptable salts thereof, and epicatechin or physiologically acceptable salts thereof are by weight ratio of 1: 2.0 to 2.1: 0.43 to 0.48. 8. The composition of claim 7, wherein the content of cyanidin-3-O-glucoside, procyanidin B2, and epicatechin, or physiologically acceptable salt thereof, is from 0.005% to 99.9% by weight of the composition. The composition of claim 7, wherein the skin health is prevention of wrinkles or improvement of wrinkles. The composition of claim 7, which is food, cosmetic, or pharmaceutical. The composition according to claim 7, comprising an extract obtained by extracting the seeds of the soybean varieties deposited with the accession number KACC 98050P to the National Academy of Agricultural Science using water, alcohols of C1-6, or mixtures thereof. A method for preventing or improving skin wrinkle development in a subject, comprising administering to the subject a composition of any one of claims 1-11. The method of claim 12, wherein said administering is applied topically to the skin.

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