WO2020009538A1 - Composition for skin health including extract of seed of new soybean cultivar scel-1 - Google Patents

Abstract

Provided are a seed of new soybean cultivar SCEL-1, a plant grown therefrom and parts of the plant, and an extract extracted from the seed.

Description

COMPOSITION FOR SKIN HEALTH INCLUDING EXTRACT OF SEED OF NEW SOYBEAN CULTIVAR SCEL-1

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of Korean Patent Application No. 10-2018-0078246, filed on July 5, 2018, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein in its entirety by reference.

Field

One or more embodiments relate to a composition for skin health, the composition including an extract of a seed of new soybean cultivar SCEL-1

Soybeans are an abundant crop source of vegetable proteins, and contain not only proteins, but also a variety of excellent functional materials including unsaturated fatty acids, amino acids, isoflavones, and phenolic acids. In this regard, soybeans are being used as a protein source to replace animal proteins.

Researchers, CHA, Baecheon et al. (The Korean Society of Pharmacognosy, Vol. 27(3): pages 190-195 (published in 1996)), discovered that an extract obtained by performing an extraction process (hereinafter referred to as an ethanol extract) on a wild-type soybean Glycine soja Siebold et Zucc. by using ethanol contains (-)-epicatechin, unlike an ethanol extract of another soybean soybean Glycine max (L.) Merrill. In addition, the researchers also discovered that the ethanol extract of the wild-type soybean has antioxidative activity. However, it is not disclosed that the soybean Glycine max (L.) Merrill contains (-)-epicatechin.

Therefore, there is a demand for a new soybean cultivar having excellent antioxidant and/or other physiological functions, compared to the known soybeans, or for a composition including an extract obtained from the new soybean cultivar.

One or more embodiments include a composition for skin health, the composition including, as an active ingredient, an extract obtained by performing an extraction process on a seed of soybean Glycine max (L.) Merrill cultivar SCEL-1 by using water, C₁-C₆ alcohol, or a mixture thereof, wherein the seed contains cyanidin-3-O-glucoside, procyanidin B2, and epicatechin, wherein an amount of the procyanidin B2 is greater than that of the cyanidin-3-O-glucoside, and a representative sample of the seed is deposited with the Korean Agricultural Culture Collection(KACC), which is an International Depository Authority(IDA) under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure under the accession number KACC 88002BP on November 8, 2018.

One or more embodiments include a composition for skin health, the composition including, as an active ingredient, cyanidin-3-O-glucoside or a physiologically acceptable salt thereof, procyanidin B2 or a physiologically acceptable salt thereof, and epicatechin or a physiologically acceptable salt thereof, at a ratio of 1: 0.8 to 2.1: 0.43 to 1.5, for example, 1: 2.0 to 2.1: 0.43 to 0.48 on a weight basis.

One or more embodiments include a method of preventing occurrence of skin wrinkles in a subject or improving skin wrinkles in a subject, or preventing occurrence of skin inflammation in a subject or improving skin inflammation in a subject, the method including administrating the composition to a subject.

Additional aspects will be set forth in part in the description which follows and, in part, will be apparent from the description, or may be learned by practice of the presented embodiments of the disclosure.

A first aspect of the present disclosure provides a composition for skin health, the composition including, as an active ingredient, an extract obtained by performing an extraction process on a seed of soybean Glycine max (L.) Merrill cultivar SCEL-1 by using water, C₁-C₆ alcohol, or a mixture thereof, wherein the seed contains cyanidin-3-O-glucoside, procyanidin B2, and epicatechin, wherein an amount of the procyanidin B2 is greater than that of the cyanidin-3-O-glucoside, and a representative sample of the seed is deposited with the Korean Agricultural Culture Collection(KACC) under the accession number KACC 88002BP on November 8, 2018.

In the seed of the present disclosure, cyanidin-3-O-glucoside and procyanidin B2 may be contained at a ratio of 1: 0.8 or more, 1: 0.8 to 5.0, 1: 0.8 to 3.0, 1: 0.8 to 2.5, or 1: 0.8 to 2.1, on a weight basis. For example, in the seed of the present disclosure, cyanidin-3-O-glucoside and procyanidin B2 may be contained at a ratio of 1: 2.0 or more, 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, on a weight basis. The term "amount" as used herein may be calculated based on an extract obtained by pulverizing the seed and incubating the pulverized seed at a temperature in a range of about 25 ℃ to about 55 ℃ for 6 hours by using 70 (v/v)% aqueous ethanol. The term "amount" as used herein is described by way of example, and should be understood as being variable depending on soil, climate conditions, and an individual seed that are considered for production of a plant body of a seed.

In the seed of the present disclosure, a total amount of procyanidin B2 and epicatechin may be, on a weight basis, twice or more or 2.5 times or more, for example, 2.6 times to 5.1 times greater than that of a cultivar named Wonheug, which is a standard cultivar.

In the composition of the present disclosure, a total amount of cyanidine-3-O-glucoside, procyanidine B2, and epicatechin in the extract may be in a range of about 2.1 % to about 3.4 % based on a total weight of the extract, and a total amount of procyanidine B2 and epicatechin in the extract may be in a range of about 1.31 % to about 2.4 % based on a total weight of the extract.

In the composition of the present disclosure, cyanidin-3-O-glucoside, procyanidin B2, and epicatechin may be contained at a ratio of 1: 0.8 to 2.1: 0.43 to 1.5, for example, 1: 2.0 to 2.1: 0.43 to 0.48 on a weight basis.

In the composition of the present disclosure, an amount of the extract may be in a range of about 0.001 % to about 99.9 %, about 0.005 % to about 99.9 %, about 0.01 % to about 80.0 %, about 0.01 % to about 60.0 %, about 0.01 % to about 50.0 %, about 0.01 % to about 30.0 %, about 0.01 % to about 20.0 %, about 0.01 % to about 15.0 %, about 0.01 % to about 10.0 %, about 0.01 % to about 5.0 %, about 0.1% to about 99.9 %, about 1.0 % to about 80.0 %, about 5.0 % to about 60.0 %, about 5.0 % to about 50.0 %, about 5.0 % to about 30.0 %, about 30.0% to about 50.0 %, about 40.0% to about 80.0 %, about 15.0 % to about 70.0 %, or about 50.0% to about 90.0 %, based on a total weight of the composition.

When the seed of the present disclosure germinates and forms a plant body, such a plant body may have characteristics listed in Table 1. Unless otherwise described in the specification, the characteristics of the soybean cultivar may be measured and verified according to "The guidelines for investigation of characteristics of each crop for examination of new varieties: Soybean Glycine max (L.) Merrill (Korea Seed Variety Service (KSVS) of the Ministry of Agriculture, Food and Rural Affairs (MAFRA), 2014: http.//www.seed.go.kr)", wherein the guidelines determine matters necessary to explain the characteristics of the varieties for each crop in Annex 1 in Article 2 of the Seed Management Guidelines pursuant to Article 30 of the Act of Protection of New Varieties of Plants and Article 33 of the Enforcement Decree of the same Act, and the directions for the qualification tests necessary for cultivation examination according to Article 47 of Enforcement Regulation of the same Act. In addition, unless otherwise described herein, quantitative traits among the characteristics of the soybean cultivar are represented as mean values.

The composition may not include any extract or active ingredient other than the extract described above, and in this regard, the extract described above may be included as the only extract or active ingredient.

A second aspect of the present disclosure provides a composition for skin health, the composition including, as an active ingredient, cyanidin-3-O-glucoside or a physiologically acceptable salt thereof, procyanidin B2 or a physiologically acceptable salt thereof, and epicatechin or a physiologically acceptable salt thereof at a ratio of 1: 0.8 to 2.1: 0.43 to 1.5, for example, 1: 2.0 to 2.1: 0.43 to 0.48 on a weight basis.

In the composition of the present disclosure, an amount of each of cyanidin-3-O-glucoside or a physiologically acceptable salt thereof, procyanidin B2 or a physiologically acceptable salt thereof, and epicatechin or a physiologically acceptable salt thereof may be in a range of about 0.001% to about 99.9 %, about 0.005% to about 99.9 %, about 0.01% to about 80.0 %, about 0.01% to about 60.0 %, about 0.01% to about 50.0 %, about 0.01% to about 30.0 %, about 0.01 % to about 20.0 %, about 0.01 % to about 15.0 %, about 0.01 % to about 10.0 %, about 0.01 % to about 5.0 %, about 0.1% to about 99.9 %, about 1.0 % to about 80.0 %, about 5.0 % to about 60.0 %, about 5.0 % to about 50.0 %, about 5.0 % to about 30.0 %, about 30.0% to about 50.0 %, about 40.0% to about 80.0 %, about 15.0% to about 70.0 %, or about 50.0% to about 90.0 %.

The composition of each of the first aspect and the second aspect of the present disclosure may be food, a cosmetic composition, or a pharmaceutical composition.

In the second aspect, the expression "physiologically acceptable salt thereof" as used herein includes a meaning of "pharmaceutically acceptable salt thereof". The term expression "pharmaceutically acceptable salt thereof" refers to possible use on animals, more particularly, to humans, without having significant toxic effects when used in a typical medicinal dosage. This expression infers that, for example, one that can be or is approved by a government or regulatory organization equivalent thereto, or one that is listed in the pharmacopoeia or recognized in other general pharmacopoeias.

In the second aspect, the expression "pharmaceutically acceptable salt thereof" refers to a salt according to an embodiment of the present disclosure being pharmaceutically acceptable and having pharmacological activity of a parent compound. Such a salt may include: (1) an acid addition salt formed of an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, or the like; or an organic acid, such as acetic acid, propionic acid, hexanoic acid, cyclopentane propionic 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, cinnamic acid, 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-en-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, or the like; or (2) a salt formed by substitution of an acidic proton in a parent compound.

In the first aspect and the second aspect, the term "active ingredient" refers to an ingredient which exhibits a desired activity alone or an ingredient capable of exhibiting activity with a carrier that is not active by itself.

In the first aspect and the second aspect of the present disclosure, the expression "for skin health" refers to preventing occurrence of skin wrinkles or improving skin wrinkles, or preventing skin inflammation or improving skin inflammation, wherein a cause of the skin wrinkles or the skin inflammation may include external stress, such as ultraviolet rays or oxidizing agents on the skin; or internal stress.

In the composition for skin health according to an aspect of the present disclosure, the composition may be also able to prevent or treat skin wrinkles and/or skin inflammation.

In the second aspect of the present disclosure, the composition may include, as an active ingredient, an extract obtained by performing an extraction process on a soybean cultivar seed by using water, C₁-C₆ alcohol, or a mixture thereof, wherein a representative sample of the soybean cultivar seed is deposited with the Korean Agricultural Culture Collection(KACC) under the accession number KACC 88002BP on November 8, 2018.

In one embodiment, the composition may be one that is effective for skin health, and more particularly, may be one that is effective for skin damage caused by ultraviolet rays or oxidative stress.

When the composition of the present disclosure is applied to medicines, the composition may be formulated into a dosage form in a solid, semi-solid, or liquid form for oral or parenteral administration by adding a commonly used inorganic or organic carrier thereto which includes the composition as an active ingredient.

Examples of the dosage form for oral administration may include tablets, pills, granules, capsules, powders, infinitesimal grains, emulsions, syrup, pellets, and the like. In addition, examples of the dosage form for parenteral administration may include injections, instillations, ointments, lotions, sprays, suspensions, emulsions, suppositories, and the like. To formulate the active ingredient of the present disclosure, the active ingredient may be easily formulated according to the conventional method, and a surfactant, an excipient, a coloring agent, a flavoring agent, a preservative, a stabilizer, a buffer, a suspension, and other common adjuvants may be used as appropriate.

The composition according to the present disclosure may be administered orally, parenterally, rectally, topically, transdermally, intravascularly, intramuscularly, intraperitoneally, subcutaneously, and the like.

In addition, a dosage of the active ingredient may vary depending on the age, gender, and weight of a subject to be treated, a particular disease or pathological condition to be treated, the severity of a disease or pathological condition, a route of administration, and a determination made by a prescriber. The determination of the dosage based on these factors may be within a level of one of ordinary skill in the art, and the dosage may be in a range of about 0.001 mg/day to about 2,000 mg/kg/day, more particularly, a range about 0.5 mg/kg/day to about 1,500 mg/kg/day.

Regarding the composition for skin health according to an aspect of the present disclosure, the composition may include a health food composition.

In one embodiment, the composition may be processed into a drink, fermented milk, a cheese, a yogurt, a juice, a probiotic agent, and a health supplement, each including the composition. In addition, the composition may be used in a variety of other food additives.

In one or more embodiments, the composition may contain other components that exhibit a synergistic effect with main effects within a range that does not damage the intended main effects of the present disclosure. For example, to improve physical properties, the composition may further include an additive, such as a flavoring agent, a dye, a bactericide, an antioxidant, a preservative, a moisturizer, an instillation, an inorganic salt, an emulsifier, or a synthetic polymer. In addition, the composition may further include an adjuvant component, such as a water-soluble vitamin, an oil-soluble vitamin, a polymeric peptide, a polymeric polysaccharide, or a seaweed extract. The components above may be appropriately selected and mixed by one of ordinary skill in the art without difficulty depending on the formulation and purpose of use, and an amount of the components to be added may be selected within a range that does not damage the objects and effects of the present disclosure.

The composition of the present disclosure may be in various forms, such as a solution, an emulsion, a viscous mixture, a tablet, a powder, and the like, and may be administered by various methods using a simple drink, an injection, a spray, or a squeezer.

The composition may include a cosmetic composition, and may be formulated in a parenteral dosage form. An example of the parenteral dosage form may include an injection or an external skin application, and examples of the external skin application may include a cream, gel, ointment, skin emulsifier, skin suspension, transdermal delivery patch, drug-containing bandage, lotion, or any combination thereof.

In the external skin application, components such as typical cosmetics or medicines used for external skin applications, for example, an aqueous component, an oily component, a powder component, an alcohol component, a moisturizer, a thickener, an ultraviolet absorber, a whitening agent, an antiseptic, an antioxidant, a surfactant, a flavoring agent, a dye, various skin nutrients, or any combination thereof, may be appropriately mixed as needed.

In the external skin application, a chelating agent such as disodium edentate, trisodium edentate, sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconic acid, and the like; caffeine, tannin, verapamil, licorice extract, glabridin, hot water extract of calines from fruit, and various herb medicines; medicines such as tocopherol acetate, glycyrrhizic acid, tranexamic acid, or a derivative or salt of any of the foregoing; vitamin C, ascorbic acid magnesium phosphate, ascorbic acid glucoside, arbutin, and kojic acid; and sugars such as glucose, fructose, and trehalose, may be mixed as appropriate.

In the composition for skin health according to an aspect of the present disclosure, the extract of the soybean cultivar SCEL-1 may be obtained by performing an extraction process on the soybean cultivar SCEL-1 by using water, C₁-C₆ alcohol, or a mixture thereof. In detail, the extract of the soybean SCEL-1 may be obtained by using methanol, ethanol, propanol, butanol, pentanol, or hexanol.

The extract of the soybean cultivar SCEL-1 of the present disclosure may include a crude extract obtained by performing an extraction process thereon by using a solvent, such as water, C₁-C₆ alcohol, or a mixture thereof. The C₁-C₆ alcohol may be, for example, methanol or ethanol. When an extraction process is performed on a soybean by using a solvent, a volume of the solvent may be about 2 times to about 15 times, about 3 times to about 15 times, about 5 times to about 15 times, or about 10 times greater than the soybean. The extraction process may include heat extraction, cold extraction, reflux cooling extraction, or ultrasonic extraction, and there is no limitation as long as the extraction process is obvious to one of ordinary skill in the art. The extraction process may be performed at room temperature, but for better efficient extraction, the extraction process may be performed under warm conditions. For example, the extraction process may be performed at a temperature in a range of about 40 ℃ to about 100 ℃, about 40 ℃ to about 80 ℃, about 40 ℃ to about 60 ℃, or at a temperature of about 50 ℃. The time for which the extraction process is performed may be in a range of about 2 hours to about 8 hours, about 4 hours to about 8 hours, about 5 hours to about 7 hours, about 5.5 hours to about 6.5 hours, or may be about 6 hours. The temperature may vary depending on conditions including a solvent used for the extraction process. To obtain a greater amount of active ingredients, the extraction process may be performed one or more times. For example, an extract obtained by combining all the extracts from the extraction process performed one to 5 times, one to 4 times, or 3 times consecutively may be used.

The extract of the soybean cultivar SCEL-1 of the present disclosure may include a crude extract of the soybean cultivar SCEL-1, and may be contained as a water-soluble fraction of the organic solvent obtained by further extraction performed on the crude extract.

Examples of the organic solvent may include hexane, methylene chloride, ethyl acetate, n-butanol, and the like. According to the method of the present disclosure, the extract or the water-soluble fraction thereof may be used as it is. In one embodiment, the extract may be used as a concentrate obtained by concentration, and in one or more embodiments, the extract may be used in a lyophilized form obtained by concentration followed by lyophilization.

A third aspect of the present disclosure provides a method of preventing occurrence of skin wrinkles in a subject or improving skin wrinkles in a subject, or preventing skin inflammation in a subject or improving skin inflammation in a subject, the method including administrating the composition of the first or second aspect to a subject.

The administrating may be oral or parenteral administration. The method according to an aspect of the present disclosure may be able to prevent occurrence of skin wrinkles in a subject or to improve skin wrinkles in a subject. The method according to an aspect of the present disclosure may be also to prevent occurrence of skin inflammation in a subject or to improve skin inflammation in a subject.

The method of the present disclosure may include administrating the composition to the skin of a subject. Here, the administrating may include coating or applying the composition to the skin. The method of the present disclosure may be considered as a make-up method, and may prevent or reduce the occurrence of reactive oxygen species (ROS) in the skin. The ROS may be produced when ultraviolet rays are irradiated to the skin.

The subject may include a mammal including a vertebrate, or fish. The vertebrate may include a cow, a pig, a cat, a dog, or a sheep. That is, the subject may be a mammal other than a human.

The above and other aspects, features, and advantages of certain embodiments of the disclosure will be more apparent from the following description taken in conjunction with the accompanying drawings, in which:

FIG. 1 is a diagram showing effects of an extract of a selected from soybean cultivar on the improvement of wrinkles of the skin damaged by ultraviolet rays in a nude mouse;

FIG. 2 shows the results of staining a tail tissue derived from a nude mouse irradiated with ultraviolet rays; and

FIG. 3 shows the results of the H&E staining performed on the mouse skin irradiated with ultraviolet rays and effects of an extract of a soybean on the collagen density in the mouse skin.

Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to like elements throughout. In this regard, the present embodiments may have different forms and should not be construed as being limited to the descriptions set forth herein. Accordingly, the embodiments are merely described below, by referring to the figures, to explain aspects of the present description. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. Expressions such as "at least one of," when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.

Examples 1: Breeding and characteristics of new cultivar

1. Breeding of cultivar

20 soybean seed grains, which are black flat-shaped granules collected as genetic resources from Idong-eup, Yongin-si, Gyeonggi-do, Korea, were prepared. As a result of cultivating the soybean seeds, separation occurred in terms of shape and color of the flowers, and in this regard, it was confirmed that the traits of the soybean seeds were not fixed. For example, the flower color appeared separately as white or pink. In this regard, the traits of the soybean seeds did not seem to be fixed, and thus, the soybean seeds were not recognized as species due to lack of stability and uniformity.

Therefore, the inventors of the present disclosure bred a cultivar by using the soybean seeds according to a pure line selection breeding method. The pure line selection was carried out from 2013 to 2017, and more particularly, it was carried out by the whole crop experimental field in the Rural Development Administration (RDA): National Institute of Crop Science (NICS) located in Suwon, Republic of Korea from 2013 to 2014, and then, in the field located in Iseo-myeon, Wanju-gun, Republic of Korea from 2015 to 2017.

A specific process of the pure line selection from the collected genetic resources of the soybean is as follows: as genetic resources, 20 soybean seeds were sown at the beginning of June every year from 2013 to 2016 in the experimental field affiliated with the RDA; then, 1 seed was selected for the purity improvement at the end of November and then harvested as a sowing seed for the following year. After four years of the pure line selection process, one finally fixed pure line seed was selected.

To confirm uniformity, traits, such as hypocotyl color, flower color, trichome color, legume color, leaf shape, growth type, flowering periods, and maturity, were examined every year. In the pure line separation process performed on the collected genetic resources, separation was observed in the hypocotyls color and the flower color in 2013, whereas no separation was observed with respect to the other traits. In addition, the progeny selected in 2013 was found to maintain uniformity in target traits among traits examined from 2014 to 2017. The distinguishable features between a standard cultivar (named Wonheug) and a control cultivar (named Cheongja-3) are clearly shown in Table 1 in terms of growth type, days to flowering, hypocotyl color, weight of 100 beans, and leaf shape. Table 1 shows the characteristics of the finally selected pure line. In Table 1, the finally selected line (cultivar) was named SCEL-1.

Source	Growth type (determinate, indeterminate)	Number of days to flowering	Flower color (violet or white)	Hypocotyl color (violet or green)	Trichome color(brown, light gray)	Testa	Hilum color	100-seed weight(g)	Leaf shape
Wonheug (standard)	Determinate	66	Violet	Violet	Brown	Black	Black	11.6	Globular
Cheongja-3 (control)	Determinate	70	Violet	Violet	Brown	Black	Black	40.7	Globular
Selected cultivar (SCEL-1)	Indeterminate	58	White	Green	Brown	Black	Black	8.8	Oblique ellipse

2. Identification of cultivar characteristics relative to control cultivar

To compare the agricultural performance of the finally selected pure line cultivar SCEL-1 relative to the existing cultivars, the performance of each cultivar was identified by repeating the randomized block design three times by using the Wonheug bean as a standard cultivar and Cheongja-3 as a control cultivar in the field affiliated with the NISC in summer of 2017. Here, the agronomic traits of each cultivar were examined by cultivating seeds after sowing two seed grains per row in an experimental plot (60 cm X 15 cm) containing four rows (4 meters long) on June 8, 2017, and then cultivating the seeds. The Wonheug bean (Cultivar Application Publication Number: 2010-341) is a microcarpa bean cultivar with a black seed coat developed by the NICS in 2009, and is currently the most cultivated cultivar in farming. The Wonheug bean is similar to the selected cultivar SCEL-1, and thus, may be used as a standard cultivar for the comparison. The control cultivar, Cheongja-3 (Cultivar Application Publication Number: 2005-176), was developed by the NICS in 2004 and is a soybean cultivar that has a black seed coat, is large-sized, and is used for cooking with rice. The Wonheug bean and Cheongja-3 are commercially available from the Korean Seed and Variety Service (KSVS) or the like.

The agronomic traits and traits examined for the performance testing were examined mainly in terms of yield component traits, such as stem length, number of nodes, number of branches, and the like. To examine such yield component traits, 10 plants with repeated nodes were examined during a maturation period in terms of stem length, number of nodes, number of branches, number of pods, 100-seed weight, yield per plant, yield per area, and yield per 10 acres (a).

Tables 2 and 3 show the characteristics of the selected soybean cultivar, the standard cultivar, and the control cultivar.

Source	Length of stem (cm)	Number of nodes	Number of branches	Seed number per pod
				1 pod	2 pods	3 pods
Wonheug (standard)	56.6±4.3	14.7±1.7	6.7±2.1	18.8±2.3	201±21	32±15.2
Cheongja-3 (control)	71.9±2.7	14.1±0.3	7.3±0.6	13.8±4.9	81±12	4.7±0.8
Selected cultivar (SCEL-1)	82.9±1.4	15.8±0.5	6.4±0.3	12.9±2.3	133±44	38.3±8.6

Source	Total number of pods	Number of grains	100-seed weight (g)	Yield per plant(g)	Yield per area(kg)	Yield per 10 acre (a)(kg)
Wonheug (standard)	252±31	512±70	11.6±0.2	32.9±3.3	6.6±0.7	587±60
Cheongja-3 (control)	100±12	191±25	40.7±3.1	26.7±3.2	5.3±0.6	477±57
Selected cultivar (SCEL-1)	184±54	397±113	9.4±0.4	23.1±2.0	4.6±0.4	413±36

Referring to Tables 2 and 3, it was found from the examination that the selected cultivar was longer than the standard cultivar, Wonheug, by 26 cm, and had one more node than the standard cultivar, Wonheug. In addition, regarding the number of branches, it was found from the examination that the selected cultivar was had about 6.4 branches similar to the standard cultivar, and regarding the number of pods, the constitution ratio of the 1-pod, 2-pods, and 3-pods was similar to the standard cultivar. Regarding the total number of pods, the number of grains, the yield per plant, and the yield per area, which indicate the yield component traits, the selected cultivar had 184 pods, 397 grains, 23.1 g of the yield per plant, and 4.6 g of the yield per area, wherein the resulting numbers and yields were lower than those of the standard cultivar. Here, 100 grains were weighed at 9.4 g, and in this regard, the selected cultivar had small grains as compared with the standard cultivar. When the yield per 10a was calculated based on the results above, the selected cultivar showed 413 kg/10 a, which was only about 70% of the yield per 10a of the standard cultivar, Wonheug. However, the amounts of procyanidin B2 and epicatechin, which are functional substances, were about 301% and 217%, respectively. In detail, the amount of procyanidin B2 was 149.4 ug/100 mg, 49.5 ug/100 mg, and 34.3 ug/100 mg in the selected cultivar SCEL-1, Wonheug, and Cheongja-3, respectively. The amount of epicatechin was 46.9 ug/100 mg, 21.6 ug/100 mg, and 20.1 ug/100 mg in the selected cultivar SCEL-1, Wonheug, and Cheongja-3, respectively. Here, the amounts of procyanidin B2 and epicatechin were confirmed by an extraction process performed according to the method of Section 1 in Example 2.[0016] According to the experiments, the cultivar SCEL-1 was found to have high uniformity and was distinguished from the standard cultivar and the existing cultivars by the comparison, and was also able to be recognized as an independent cultivar with distinct characteristics in terms of the agronomic traits, the yield ability, and the functional material contents.

This newly selected cultivar of a black small-sized bean having an oblique ellipse shape was named SCEL-1, and was deposited with the Korean Agricultural Culture Collection(KACC) under the accession number KACC 88002BP on November 8, 2018. FIG. 1 shows a schematic breeding diagram of a soybean cultivar according to the present disclosure.

Example 2: Extract obtained by performing an extraction process on a newly selected soybean seed by using water, alcohol, or a mixture, and use of the extract

In this Example, an extract was prepared from a seed of the newly selected soybean cultivar SCEL-1 of Example 1, and an effect of the extract was identified.

1. Preparation of an extract

Immediately before analyzing a seed of the selected cultivar, the seed was pulverized by using a high-speed pulverizer (Wonder Blender, 820W, 30000 RPM, Sanplatec Corp) to prepare powdered seed. 1 g of the powdered seed thus obtained was mixed with 100 ml of 70 (v/v)% aqueous ethanol in a glass tube, and the mixed solution was stirred at a temperature of 50 ℃ for 6 hours by using a magnetic bar. Then, the resulting extract was filtered through filter paper (Garde No.131 Qualitative filter paper, Advantec).

Afterwards, the ethanol layer was dried in a nitrogen gas drier (hurricane-Eagle, Chongmin Technology) for 1 hour, and then, was lyophilized for one day to completely remove moisture,thereby obtaining 0.19 g (standard deviation SD: ±0.015) of a soybean extract. In the experiments, for use of the soybean extract, it was dissolved at a concentration of 30 mg/ml in 50% v/v ethanol, and was subjected to the HPLC analysis.

2. Confirmation of wrinkle improving effect

By using the extract obtained in Section 1, the efficacy of the extract of improving wrinkles damaged by ultraviolet rays was confirmed through an experiment using the EpiDermFT^TM 3D skin product and a nude mouse model.

(1) Confirmation of using artificial skin

In detail, the EpiDermFT^TM 3D skin product manufactured by MatTek Company was purchased and used in this experiment. After a 6-well plate was filled with 900 ul of the medium provided by the manufacturer, the prepared 3D skin product was placed on each well of the plate, and then, cultured in an incubator under the conditions of 37 ℃, 5 % CO₂, and 100 % humidity. After 24 hours, ultraviolet B (UVB) rays (40 mJ/cm²) were irradiated, and the medium in each cell was replaced with a medium containing test materials in an amount of 10 ug/ml, 30 ug/ml, and 100 ug/ml, respectively, After 72 hours of incubation, the culture medium at the bottom, i.e., the culture medium containing collagenase (e.g., MMP-1) and procollagen that were secreted from the artificial skin product was harvested. Then, the amount of type I collagenase (e.g., MMP-1) produced was quantified using a method according to the ELISA kit (AP biotech RPN2610). In addition, the same supernatant was used to quantify the amount of residual, undegraded procollagen using a method according to the ELISA kit (Takara MK101). Each quantified value was expressed as the amount of protein per ml, And the results of the quantification are shown in Table 4 and FIG. 1.

Table 4 and FIG. 1 show the effect of the extract of the soybean of the present disclosure on the amounts of MMP-1 and procollagen in the artificial skin irradiated with ultraviolet rays.

Sample	Concentration (ug/ml)	MMP-1 biosynthesis (pg/ml)	Amount of residual procollagen
Untreated group		3980 ± 250	19000 ± 320
UVB-induced group(40 mJ/cm2)		11800 ± 300	15000 ± 220
Soybean SCEL-1	10	9980 ± 220	17000 ± 130
	30	8100 ± 230	19000 ± 260
	100	7600 ± 180	22000 ± 379
Black soybbean	10	11200 ± 350	16000 ± 400
	30	10400 ± 320	17300 ± 480
	100	8500 ± 350	18500 ± 500
Rhynchosia nulubilis	10	10800 ± 340	16800 ± 496
	30	10100 ± 380	18100 ± 550
	100	8300 ± 220	19200 ± 460

Referring to Table 4 and FIG. 1, it was confirmed that, by treating the extract of the soybean cultivar SCEL-1, the biosynthesis of matrix metalloprotease (MMP-1), which is type I collagenase caused by UVB, was reduced. Accordingly, it was also confirmed that the degradation of procollagen was prevented. As a result of comparing three beans, it was confirmed that soybean cultivar SCEL-1 showed significantly excellent effects compared to other types of black beans.

(2) Confirmation of using an animal model

When orally administering the extract obtained in Section 1, the efficacy of the extract of improving skin wrinkles was confirmed through an experiment using a nude mouse model.

8-week-old nude mice (OrientBio, SKH-1 species, and average weight of 26.2 g) were divided into two groups, each having seven mice. Here, one group is a control group, and the other group is a test group. In the test group, based on a weight, 2.5 mg/10 ml and 5.0 mg/10 ml of an extract solution were each administered to a mouse to be 25 mg/kg/day and 50 mg/kg/day, respectively, wherein the extract solution was obtained by dissolving the lyophilized extract of Section 1 in an aqueous solution containing 2 % ethanol, 1 % Tween 80, and 0.8 % carboxymethyl cellulose (CMC). In the control group, based on a volume, an aqueous solution containing 2 % ethanol, 1 % Tween 80, and 0.8 % CMC was administered to a mouse. Here, the administration was performed by direct administration to the stomach of the mouse using an oral zoned. In this regard, the soybean extract was able to be administered to the mouse at doses of 25 mg/kg/day and 50 mg/kg/day. After this administration, ultraviolet rays were irradiated to the mouse.

Along with the administration of the extract solution or 2 % ethanol, 1% Tween 80, and 0.8 % CMC aqueous solution, to individual mice in each of the control group and the test group, 1 minimal erythema dose (MED) of ultraviolet ray was irradiated three times a week for two weeks, 2 MEDs of ultraviolet ray was irradiated three times a week for two weeks, 3 MEDs of ultraviolet ray was irradiated twice a week for two weeks, and 4 MEDs of ultraviolet ray was irradiated twice a week for two weeks. That is, the irradiation was performed for a total of eight weeks. After oral administration of each sample once a day for 8 weeks, a skin replica was manufactured using silicone (SILFLO impression material, Flexico, Tokyo, Japan). Then, the state of wrinkles was measured using a fine wrinkle measuring meter (Visioline VL650, Courage + Khazaka, Koln, Germany) to observe the wrinkle pattern, and the wrinkle-related factor values were identified.

Among the wrinkle-related factor values, the mean form factor is calculated from the following formula with the most accurate and reliable measurement method. The mean form factor is represented by analyzing the depth and length of the shadows of the wrinkles created by irradiating the skin replica with the light source.

Mean form factor = 1-(Wrinkle depth/wrinkle length). Here, this value is always between 0 and 1, and the closer the measured value is to 1, the better the wrinkle improving effect is meant. The results are shown in FIG.2. FIG. 2 is a diagram showing the effect of the extract of the soybean of the present disclosure on the wrinkle pattern and the winkle-related mean form factor in the artificial skin irradiated with ultraviolet rays. Referring to FIG .2, it was confirmed that the group to which the soybean cultivar SCEL-1 was orally administered at a dose of 50 mg/kg/day had the mean form factor value of 0.7, indicating that the wrinkle-improving effect was excellent compared to other test groups treated with other soybeans.

In addition, the skin tissue of the nude mouse after the end of the experiment was treated with formalin, and to observe the thickness of the skin tissue, the skin tissue was subjected to the H&E staining. The results are shown in Table 5. Also, to observe changes in collagen, Masson Trichome staining was performed on the skin tissue, and the results are shown in Table 6 and FIG. 3. FIG. 3 shows the results of the H&E staining performed on the mouse skin irradiated with ultraviolet rays and effects of the extract of the soybean of the present disclosure the collagen density in the mouse skin.

Sample	Oral dose (mg/kg/day)	Epidermal thickness
Untreated group		15.5 ± 0.84
UVB (40 mJ/cm2)		55.2 ± 0.08
Soybean SCEL-1	25	46.5 ± 3.35
	50	43.9 ± 1.19
General black soybean	25	55.6 ± 0.98
	50	55.7 ± 1.39
Rhynchosia nulubilis(Wonju)	25	51.9 ± 2.17
	50	55.0 ± 0.38

Referring to Table 5, a test group treated with the soybean SCEL-1 showed an effect of preventing the skin from thickening by referring to that the epidermal thickness of the skin was significantly small upon ultraviolet irradiation.

Sample	Oral dose (mg/kg/day)	Amount of collagen (%)
Untreated group		193.8 ± 5.6
UVB (40 mJ/cm2)		100 ± 7.1
Soybean SCEL-1	25	147.6 ± 11.5
	50	176.1 ± 6.8
Black soybean	25	150.7 ± 5.1
	50	161.0 ± 7.2
Rhynchosia nulubilis	25	147.3 ± 4.7
	50	159.2 ± 7.2

Referring to FIG. 3 and Table 6, a test group treated with the soybean SCEL-1 was superior to other types of beans in preventing the collagen breakage during ultraviolet irradiation and restoring the collagen activity.[0041] From the results above, it was confirmed that the soybean SCEL-1 extract showed excellent efficacy for the improvement of skin tissue or wrinkles damaged by ultraviolet rays.

3. Analysis of components of soybean extract

The soybean extract was subjected to mass spectrometry, so as to analyze active ingredients in the soybean extract.

In detail, the analysis of components in the soybean extract was carried out by using an Agilent 1260 HPLC system and a Bruker MicrOTOF-Q II mass spectrometer. Here, for the column analysis, a Prevail C18 column (250 mm x 4.6 mm, 5 um), Solvent A of a mobile phase consisting of 95% water/5% acetonitrile (0.1% formic acid), and Solvent B of a mobile phase consisting of 95% acetonitrile/5% water (0.1% formic acid) were used. Here, the flow rate of the solvent was set at 0.7 ml/min, and the concentration gradient conditions of the solvent used for the separation of components are shown in Table 7.

Time (min)	A(%)	B(%)	Flow rate (ml/min)	Maximum 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

Here, the temperature of the column was maintained at 35 ℃, and 10 μL of the sample was injected into the column. A mass spectrometer was used to analyze ingredients contained in the soybean extract under conditions of mode: ESI(+); mass range: 50 m/z to 800 m/z; nebulizing gas: 8 L/min; source gas temperature: 180 ℃; capillary voltage: +4,500 V; and cone voltage: 35 V.

As a result, the amount of each of epicatechin and procyanidin B2 contained in the selected cultivar named SCEL-1 was in a range of about 0.32% to about 0.46% and in a range of about 1.0% to about 2.0%, respectively, which was significantly higher than the amounts thereof in Wonheug, which were respectively in a range of about 0.14% to about 0.15 % and in a range of about 0.33% to about 0.35%.

In cultivar named SCEL-1 selected on the basis of cyanidin-3-O-glucoside which is a representative substance of anthocyanin antioxidants, cyanidin-3-O-glucoside, procyanidin B2, and epicatechin were contained at a ratio of 1: 0.8 to 2.1: 0.43 to 1.5, for example, 1: 2.0 to 2.1: 0.43 to 0.48. Meanwhile, in Wonheug, cyanidin-3-O-glucoside, procyanidin B2, and epicatechin were contained at a ratio of 1: 0.43 to 0.70 : 0.19 to 0.25.

It should be understood that embodiments described herein should be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each embodiment should typically be considered as available for other similar features or aspects in other embodiments. While one or more embodiments have been described with reference to the figures, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the disclosure as defined by the following claims.

According to the one or more embodiments, a composition for skin health, the composition including an extract of soybean cultivar SCEL-1, may be used to prevent or improve skin wrinkles and/or skin inflammation.

According to the one or more embodiments, a method of preventing occurrence of skin wrinkles in a subject or improving skin wrinkles in a subject, or preventing occurrence of skin inflammation in a subject or improving skin inflammation in a subject may be used to prevent occurrence of skin wrinkles in a subject or improve skin wrinkles in a subject, or to prevent skin inflammation in a subject or improve skin inflammation in a subject.

Claims (13)

1. A composition for skin health, the composition comprising, as an active ingredient,

   an extract obtained by performing an extraction process on a seed of soybean Glycine max (L.) Merrill cultivar SCEL-1 by using water, C₁-C₆ alcohol, or a mixture thereof, wherein the seed contains cyanidin-3-O-glucoside, procyanidin B2, and epicatechin, wherein an amount of the procyanidin B2 is greater than that of the cyanidin-3-O-glucoside, and a representative sample of the seed is deposited with the Korean Agricultural Culture Collection(KACC) under the accession number KACC 88002BP on November 8, 2018.
2. The composition of claim 1, wherein a total amount of cyanidine-3-O-glucoside, procyanidine B2, and epicatechin in the extract is in a range of 2.1 % to 3.4 % based on a total weight of the extract.
3. The composition of claim 2, wherein the extract contains cyanidin-3-O-glucoside, the procyanidin B2, and the epicatechin at a ratio of 1: 0.8 to 2.1: 0.43 to 1.5 based on a weight basis.
4. The composition of claim 1, wherein an amount of the extract is in a range of 0.005 % to 99.9 % based on a total weight of the composition.
5. The composition of claim 1, wherein the skin health includes prevention of skin wrinkles or improvement of skin wrinkles.
6. The composition of claim 1, wherein the composition is food, a cosmetic composition, or a pharmaceutical composition.
7. A composition for skin health, the composition comprising, as an active ingredient,

   cyanidin-3-O-glucoside or a physiologically acceptable salt thereof, procyanidin B2 or a physiologically acceptable salt thereof, and epicatechin or a physiologically acceptable salt thereof at a ratio of 1: 0.8 to 2.1: 0.43 to 1.5 on a weight basis, respectively.
8. The composition of claim 7, wherein an amount of each of the cyanidin-3-O-glucoside or a physiologically acceptable salt thereof, the procyanidin B2 or a physiologically acceptable salt thereof, and the epicatechin or a physiologically acceptable salt thereof is in a range of 0.005 % to 99.9 %.
9. The composition of claim 7, wherein the skin health includes prevention of skin wrinkles or improvement of skin wrinkles.
10. The composition of claim 7, wherein the composition is food, a cosmetic composition, or a pharmaceutical composition.
11. The composition of claim 7, wherein the composition comprises an extract obtained by performing an extraction process on a seed of soybean cultivar by using water, C₁-C₆ alcohol, or a mixture thereof, wherein a representative sample of the soybean cultivar seed is deposited with the Korean Agricultural Culture Collection(KACC) under the accession number KACC 88002BP on November 8, 2018.
12. A method of preventing occurrence of skin wrinkles in a subject or improving skin wrinkles in a subject, the method comprising

    administering the composition of claim 1 to a subject.
13. The method of claim 12, wherein the administration is applied locally to the skin.

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