KR102457475B1 - Cosmetic and functional food composition comprising caffeoylmalic acid having whitening and anti-photoaging activities - Google Patents

Abstract

The present invention relates to a skin whitening and photoaging inhibitory cosmetic composition and cosmetic food containing caffeoyl malic acid. The composition according to the present application exhibits excellent whitening and photoaging inhibitory effects through inhibition of melanin production and migration to keratinocytes, and does not show side effects due to natural origin, so it is useful as a cosmetic composition and beauty food having whitening and photoaging inhibition functions can be used

Description

Cosmetic or functional food composition comprising caffeoylmalic acid having whitening and anti-photoaging activities showing skin whitening and photoaging inhibitory activity

The present invention relates to a whitening and photoaging inhibitory cosmetic composition and cosmetic food containing a soybean extract.

In the cosmetic field, one of the fields of greatest interest to East Asian women, especially women from Korea, Japan and China, is skin whitening and anti-aging.

The most important factor in skin whitening is skin melanin pigment. Whitening functional cosmetics have the function of helping skin whitening by thinning the color of melanin pigment already deposited on the skin. In order to develop whitening functional cosmetics, functional raw materials with various mechanisms are being developed, and raw materials with a tyrosinase inhibition mechanism such as arbutin and kojic acid are the most representative.

Melanin or pigment is responsible for the color of skin, hair and eyes, and epidermal melanocytes in the skin produce melanin from melanosomes, which are specific organelles related to lysosomes (Raposo and Marks, 2002, Traffic 3, 237-248). Melanosomes progress through well-defined sequential morphological stages: the first stage is a free melanosome without melanin similar to the internal body structure, the second stage melanosome is an elongated structure with intralumenal fibres (stripes), three Stage 4 melanosomes show melanin deposition and stage 4 melanosomes leading to black streaks become filled with melanin (Raposo et al., 2001, J. Cell Biol. 152, 809-824; Seiji et al., 1963, Nature 197, 1082-1084).

In epidermal melanocytes, melanosomes undergo actin-dependent transport to microtubules and periphery before migrating to surrounding keratinocytes (Anister et al., 2002, Am. J. Hum. Genet. 71, 407-414; Marks et al., Nat. Rev. Mol. Cell Biol. 2, 738-748; Westbroek et al., 2001, Pigment Cell Res. 14, 320-327) wherein Rab27a is a lysosomal-associated organelle (LRO) in certain cell types. It acts as a member of the small GTPase Rab27 subfamily that controls transport and exocytosis (Izumi et al., 2003, Cell. Struct. Funct. 28, 465-474). Rab27a is located on the melanosome membrane in a state of binding to activated GTP, where it acts as a receptor for the actin-dependent MyosinVa motor protein mediated by melanophilin/Slac2-a, a melanocyte-specific Rab27a effector (Fukuda et al. ., 2002, J. Biol. Chem. 277, 12432-12436; Strom et al., 2002, J. Biol. Chem. 277, 25423-25430; 2003; Wu et al., 2002, Mol. Biol. Cell 13 , 1735-1749). Therefore, inhibition of Rab27a expression leads to loss of function of the triple complex of Rab27a-melanophilin-myosinVa, resulting in a defect in normal actin-dependent melanosome transport and entrapment by peripheral actin, which affects skin color.

The raw material for the mechanism of inhibiting tyrosine expression and melanosome migration is announced as a whitening functional main ingredient. Hydroquinone, a synthetic compound that inhibits melanin production, was used as a main ingredient in whitening cosmetics and was designated as a carcinogen. The core ingredients of major functional cosmetics, such as arbutin and mulberry extract, are being replaced with natural ingredients.

Korean Patent Application Laid-Open No. 2013-0001027 (published on 01/03/2013) discloses a cosmetic composition for whitening containing an extract of wild herbs and a method for preparing the same.

However, there are still unidentified various components in natural products, and there is a possibility of discovering new substances and greatly expanding their uses. Therefore, it is necessary to discover natural substances that affect the fundamental mechanisms related to the generation and movement of melanosomes.

An object of the present application is to provide a whitening and photoaging inhibitory cosmetic composition and food derived from a natural substance that can effectively inhibit melanosome migration and generation and photoaging without toxicity.

In one aspect, the present application provides a cosmetic composition for skin whitening and photoaging inhibition comprising caffeoyl malic acid.

The composition according to the present application is quercetin 3-O-beta-(2(G)-O-beta-xylopyranosyl-6(G)-O-alpha-rhamnopyranosyl)glucopyranoside) or rutin (2-(3,4-dihydroxyphenyl)- 5,7-dihydroxy-3-[α-L-rhamnopyranosyl-(1→6)-β-D-glucopyanosyloxy]-4H-chromen-4-one) may be further included.

Skin whitening by the cosmetic composition according to the present application inhibits the movement of melanin produced in melanocytes constituting the epidermal cells of the skin to keratinocytes; And it is due to the mechanism of inhibiting melanin production in melanin-forming cells, and it can be effectively used for skin whitening by effectively inhibiting the skin deposition of melanin.

In addition, in one embodiment, the inhibition of the migration of melanin into keratinocytes by the composition according to the present application is due to inhibition of the expression of Rab27A and MyosinVa involved therein, which effectively inhibits the deposition of melanin on the skin and thus can be effectively used for skin whitening. indicates.

In addition, the composition according to the present application inhibits photoaging by inhibiting the expression of cyclooxygenase-2 (COX-2), and can effectively inhibit photoaging at the molecular level of cells.

In another aspect, the present application also provides a functional food composition for improving skin whitening and photoaging, comprising caffeoyl malic acid.

In one embodiment, the functional food composition is quercetin 3-O-beta-(2(G)-O-beta-xylopyranosyl-6(G)-O-alpha-rhamnopyranosyl)glucopyranoside) or rutin (2-(3,4 -dihydroxyphenyl)-5,7-dihydroxy-3-[α-L-rhamnopyranosyl-(1→6)-β-D-glucopyanosyloxy]-4H-chromen-4-one).

Skin whitening by the food composition according to the present application inhibits the movement of melanin produced in melanocytes constituting the epidermal cells of the skin to the keratinocytes; And it is due to the mechanism of inhibiting melanin production in melanin-forming cells, and it can be effectively used for skin whitening by effectively inhibiting the skin deposition of melanin.

In addition, in one embodiment, the inhibition of the migration of melanin into keratinocytes by the food composition according to the present application is due to the inhibition of the expression of Rab27A and MyosinVa involved therein, which effectively inhibits the deposition of melanin on the skin and thus can be effectively used for skin whitening indicates

In addition, the inhibition of photoaging by the food composition according to the present application is by inhibition of COX-2 (cyclooxygenase-2) expression, and can effectively inhibit photoaging at the molecular level of cells.

1 is a photograph of green beans from which an extract according to the present application is derived, and three indicator components (caffeoylmalic acid (#1), Quercetin 3-O-beta-(2(G)-O-beta-xylopyranosyl Chemical structural formula representing -6(G)-O-alpha-rhamnopyranosyl)glucopyranoside (#2) and rutin (#3)).
2 is a result showing the effect of inhibiting the expression of Rab27A, a factor related to melanosome migration of caffeoyl malic acid isolated and identified from green beans according to one embodiment of the present application.
3 is a result showing the effect of inhibiting the expression of MyosinVa, a factor related to the melanin movement of caffeoyl malic acid isolated and identified from green beans according to one embodiment of the present application.
Figure 4 is a result showing the melanin formation inhibitory effect of caffeoylmalic acid (caffeoylmalic acid) isolated and identified from the green bean leaf extract according to one embodiment of the present application.
5 is a caffeoylmalic acid (#1), Quercetin 3-O-beta-(2(G)-O-beta-xylopyranosyl-6 (G)-O- Results showing the effect of alpha-rhamnopyranosyl)glucopyranoside (#2) and rutin (#3) on COX-2 expression induced by UV irradiation.
Figure 6a is a schematic diagram showing melanosome migration, transfer, and turnover that has been previously studied (Hume et al., 2001, The J. of Cell Biol. 152; 795-808).
Figure 6b is a schematic diagram showing the mechanism of suppression of the expression of MyosinVa, Rab27A, which are marker factors involved in the migration of melanosomes involving caffeoylmalic acid isolated and identified from the bean extract of the present application to epidermal cells (Briganti, S., et al. ., (2003) Pigment Cell Res. 16, 101-110.).

The present application is based on the discovery that caffeoylmalic acid can be used as an effective whitening and photoaging inhibiting cosmetic composition and cosmetic food because it can inhibit melanosome migration and generation and photoaging while being nontoxic.

Accordingly, in one aspect, the present application relates to a cosmetic composition for skin whitening and/or photoaging inhibition comprising caffeoyl malic acid as an active ingredient.

In one embodiment, the caffeo oil malic acid is isolated and identified from soybeans ( Canavalia gladiata ).

Green bean ( Canavalia gladiata ) is an annual creeper plant in the legume family, which flowers in June-July, produces fruit pods in August-October, and ripens in late autumn. It is native to the tropical regions of Southeast Asia, and the fruit is in the shape of a bow and the shape is similar to that of a bean sprout. 1 is a chemical structural formula showing a photograph of green beans and three index components of a green bean leaf. Soybeans contain medicinal ingredients such as urease, hemaglutinine, canavanine, canavalia gibberellin I and II, and in folk remedies, it has been used as a special drug to treat purulent inflammation such as sinusitis, hemorrhoids, and boils. Based on these excellent functions, a drink called “Evolution 851,” which was developed by fermenting soybeans in China, was introduced as a health supplement in Korea. The health tea "Dodu Power" made was also sold as a product. Although there are 65 studies reported from Canavalia gladiata worldwide, there are no studies on whitening efficacy and mineralization inhibition through the inhibition of melanin migration, which is the content of this application.

In one embodiment, the composition according to the present application may further include one or more of the following active ingredients: quercetin 3-O-beta-(2(G)-O-beta-xylopyranosyl-6(G)-O-alpha -rhamnopyranosyl)glucopyranoside) or rutin (2-(3,4-dihydroxyphenyl)- 5,7-dihydroxy-3-[α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranosyloxy]-4H-chromn- 4-one).

The skin epidermal cells include keratinocytes and melanin-forming cells (see FIG. 6a), and the active ingredient, particularly caffeoyl malic acid, isolated herein inhibits the production of melanin in the melanin-forming cells, as well as the keratinocytes of the produced melanin. inhibit the movement of

Accordingly, in one embodiment, skin whitening is by inhibition of the synthesis of melanin in melanocytes.

In another embodiment, skin whitening by the composition according to the present application is by inhibition of the migration of melanin produced in melanogenic cells constituting epidermal cells to keratinocytes.

In another embodiment, skin whitening is a gene involved in the movement of melanin to keratinocytes, Rab27A (Ras-related protein) (Jordens et al ., 2006, Pigment Cell Res. 19; 412-423) and Myosin Va ( Yao et al ., Scientific Reports | 5:10874 | DOI: 10.1038/srep10874 1) by suppression of expression. In particular, caffeoyl malic acid according to the present application inhibited the expression of Rab27A and MyosinVa, which are markers related to the migration of melanosomes (see FIGS. 2 and 3 ), which are steps I, II, and III of the skin pigmentation process ( FIGS. 6A and 6B ). See), which affects the movement of melanosomes to epidermal cells after melanin biosynthesis.

In addition, caffeoyl malic acid according to the present application is shown to inhibit the synthesis of melanin (see FIG. 4 ), and the composition according to the present application can be effectively used for skin whitening by inhibiting the synthesis and migration of melanin in epidermal cells.

In addition, the composition comprising the active ingredient according to the present application exhibits skin photoaging inhibitory efficacy. A new concept of “Inflammaging” is emerging as a compound word of “Inflammatory” and “aging” in relation to skin aging. This is presumed to be related to aging due to the inflammatory response induced by light stress such as UV. The UVB-induced inflammatory response involves the production of inflammatory mediators such as prostaglandin and leukotriene, and the production of these eicosanoids under UVB exposure conditions is closely related to the increase in the expression or activity of phospholipase A2 (PLA2). However, according to a recent study report, it was reported that the expression of COX-2 (Cyclooxygenase-2), a sub-step of PLA2 in prostaglandin production, also plays a very important role in the increase in prostaglandin production caused by UV irradiation (Lee et al . ., 2012, Exp. Mol. Med. 44; 536-544; Afnan et al ., 2016, Exp. Dermatol. 25; 440-446) . Therefore, inhibition of COX-2 expression is related to inhibition of photoaging. Herein, it was shown that the pea extract according to the present application inhibits the expression of COX-2, which is one of these inflammaging targets (see FIG. 5 ), indicating the photoaging inhibitory effect of the pea extract according to the present application.

Accordingly, the composition according to the present application can be effectively used for skin whitening and/or photoaging inhibition/improvement.

As used herein, "skin whitening" refers to suppression of changes in skin color or pigmentation caused by excessive production of pigments in the skin due to external environmental changes such as ultraviolet rays, stress, abnormal hormone secretion and pregnancy, etc. It includes an effect of improving or preventing or preventing darkening of the skin, freckles, freckles, and dark circles, an effect of increasing the translucency of the skin, and an effect of improving the dullness of the skin to increase the gloss and tightness.

In general, the pigmentation of the skin is stimulated by ultraviolet rays or hormonal imbalance, and melanocytes are stimulated, and the skin deposition of melanin pigment biosynthesized here is the main cause. Therefore, the composition according to the present application capable of inhibiting the movement of melanin from melonocytes, which are melanin biosynthetic cells constituting epidermal cells, to keratinocytes, which are other cells constituting epidermal cells, requires inhibition of pigmentation in addition to the above-mentioned whitening. It can be used for a variety of purposes.

Photoaging is one of the causes of skin aging and refers to skin aging caused by light including ultraviolet rays. Skin aging can be broadly divided into two cases, natural aging and photoaging, and is characterized by wrinkles on the skin. Wrinkles of the skin are known to be caused by a decrease in elastic fibers such as collagen or elastin due to a decrease in cell regeneration ability, the generation of lipid peroxide by free radicals, degeneration due to the oxidation of biomaterials, and fatigue due to excessive muscle exercise. . As mentioned above, the green soybean extract according to the present application may be usefully used to suppress and/or improve skin aging due to photoaging by inhibiting the expression of COX-2, one of the inflammaging targets.

As used herein, the term cosmetic composition refers to an article used in the human body to brighten the appearance by cleaning and beautify the human body, or to maintain or promote health of skin and hair, and has a slight action on the human body. In a conventional sense, it includes lotions, creams, oils, cleaning products and functional cosmetics. Functional cosmetics refers to a group of cosmetics that have added added value to act and express the functions of the cosmetics for a specific purpose using physical, biochemical, and bioengineering methods, etc. Includes cosmetics designed and processed to sufficiently express functions in the living body.

The cosmetic composition or cosmetic according to the present application may be provided as, for example, a cream, lotion, ampoule, skin, essence, soap, etc., and in another aspect, the basic cosmetic composition (lotion, cream, essence, cleansing foam and cleansing water such as face wash, pack, body oil), color cosmetic composition (foundation, lipstick, mascara, makeup base), or in the form of a mask or pack.

The portion to which the cosmetic composition or cosmetics according to the present application can be applied is not limited to a specific adipose tissue of the body, and for example, it can be applied to the skin of various areas requiring pigmentation suppression or whitening, particularly the face, arms, legs, and the like. Administration may be carried out at the site of application by various routes of administration, for example systemic or topical, particularly topical, transdermal, or injection, particularly transdermal.

The cosmetic composition according to the present application is not particularly limited in other ingredients except for including the bean extract of the present application or at least one active ingredient isolated therefrom as an essential ingredient in the indicated ratio, and in the case of conventional cosmetics, for example, a cream, vegetable oils, emulsifiers, thickeners, fragrances, water, antioxidants, and UV absorbers.

The content of the extract included in the composition according to the present application may vary depending on the specific use or formulation of the desired product, but may be included in an amount of about 0.001 to about 50% by weight in the total composition, but does not exclude those outside this range .

The cosmetic composition according to the present disclosure may include conventional adjuvants and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments and/or fragrances in addition to the soybean extract and/or one or more active ingredients isolated therefrom. have.

The cosmetic composition of the present disclosure may include an unsaturated fatty acid including conjugated linoleic acid, linoleic acid, gamma-linoleic acid, alpha-linoleic acid, or a water-soluble base material including propylene glycol.

In addition, the cosmetic composition of the present application may be prepared in any formulation conventionally prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing , oil, powder foundation, emulsion foundation, wax foundation, spray, etc., but is not limited thereto. More specifically, it may be prepared in the form of a flexible lotion, a nourishing lotion, a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a mask, a pack, a spray, or a powder.

When the formulation of the cosmetic composition of the present application is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide is used as a carrier component. can be

When the formulation of the cosmetic composition of the present application is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydrocarbon, propellants such as propane/butane or dimethyl ether.

The cosmetic composition according to the present disclosure may further comprise a cosmetically/dermatologically acceptable carrier. "Cosmetic/dermatologically acceptable" means suitable for use in contact with tissue (eg, skin or hair) without undue toxicity, incompatibility, instability, irritation, allergic reaction, etc.

When the formulation of the present composition is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 fatty acid esters of ,3-butylglycol, glycerol aliphatic esters, polyethylene glycol or sorbitan may be used.

When the formulation of the present composition is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, and microcrystals Adult cellulose, aluminum metahydroxide, bentonite, agar, or tracanth may be used.

When the formulation of the composition of the present application is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolium derivative, methyltaurate, sarcosinate, fatty acid amide ether as carrier components Sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester may be used. According to a preferred mode of administration, the composition of the present invention may comprise at least one pharmaceutically acceptable excipient, in particular a dermatologically acceptable excipient.

In addition, in the composition for skin of each formulation, other components other than the above-mentioned essential components can be selected and formulated without difficulty by those skilled in the art according to other skin compositions or purposes of use.

Since the composition of the present invention has almost no toxicity and side effects, it can be safely used even for long-term use for prophylactic purposes.

Accordingly, the green bean extract according to the present application can be used as a main, auxiliary material, and food additive of food, or as a material for health functional food such as whitening and/or photoaging improvement functional food.

As used herein, the term "food" means a natural product or processed product containing one or more nutrients, and preferably means a state that can be directly eaten through a certain processing process, In a sense, it is intended to include all foods, food additives, functional foods and beverages.

As used herein, the term "functional food" refers to a food group or food composition that has added value to act and express the function of the food for a specific purpose using physical, biochemical, and bioengineering methods, etc. , refers to foods designed and processed to sufficiently express body control functions related to disease prevention and recovery, etc. to the living body, and in particular includes "health functional foods".

As used herein, the term "health supplement" refers to a food manufactured and processed using raw materials or ingredients having useful functions in the human body, and is useful for health purposes such as regulating nutrients or physiological action with respect to the structure and function of the human body. It refers to food manufactured and processed by extracting, concentrating, refining, or mixing ingredients contained in food raw materials or using specific ingredients as raw materials for the purpose of health supplements to obtain an effect.

The functional food and health supplement may further include a food supplementary additive that is pharmaceutically acceptable, and may further include an appropriate carrier, excipient and diluent commonly used in the manufacture of functional food.

Foods to which the green bean extract according to the present application and/or one or more active ingredients separated therefrom can be added include, for example, various foods, beverages, gum, tea, vitamin complexes, functional foods, and the like. In addition, in the present invention, food includes special nutritional food (eg, formula milk, infant food, etc.), processed meat products, fish meat products, tofu, jelly, noodles (eg, ramen, noodles, etc.), health supplements, seasoned foods ( Ex, soy sauce, soybean paste, red pepper paste, mixed soy sauce, etc.), sauces, sweets (eg snacks), dairy products (eg fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various kimchi, pickles, etc.), beverages ( Examples include, but are not limited to, fruit and vegetable beverages, soy milk, fermented beverages, etc.) and natural seasonings (eg, ramen soup, etc.). The food, beverage or food additive may be prepared by a conventional manufacturing method.

In one embodiment, it is manufactured as a functional beverage, and a functional beverage is a beverage group or beverage composition with added value added so that the beverage can act and express the function of the beverage for a specific purpose using physical, biochemical, or bioengineering methods, etc. It refers to a beverage designed and processed to sufficiently express the body control functions related to defense rhythm control, disease prevention and recovery, etc. to the living body, and in particular, it refers to a beverage for improving skin elasticity and promoting collagen synthesis.

The food composition of the present application contains various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. These components may be used independently or in combination.

Hereinafter, examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the present invention is not limited thereto.

Example

Example 1. Green bean leaf ( Canavalia gladiata ) Separation and identification of the indicator compound from the extract

In order to isolate the active ingredient compound from the soybean leaf extract, an extract was prepared and a solvent fraction was prepared. Specifically, 10 kg of dried green bean leaves were extracted in 30% (W/W) aqueous ethanol solution (1→30) at 60° C. for 5 hours, then filtered with a 250 mesh filter cloth and filtered with a 0.3 μm filter. Then, the filtrate was concentrated under reduced pressure to prepare a crude extract. In order to prepare a solvent fraction, the crude extract was suspended in distilled water, and the same amount of ethyl acetate solvent was added thereto, followed by extraction twice. Thereafter, the distilled water layer was recovered and concentrated under reduced pressure to obtain a distilled water soluble fraction of green bean leaves. The distilled water soluble extract of green bean leaf was obtained by performing HP-20 resin column chromatography under the conditions of Water:EtOH (Water 100% → EtOH 30%) to divide the extract into three major fractions (CG-1 to CG-3). A single component was purified from the CG-1 fraction, and as a result of identifying the structure using a spectroscopic method, Caffeoylmalic acid (2-[(E)-3-(3,4-dihydroxyphenyl)prop-2) having the structure of Formula 1 below -enoyl]oxybutanedioic acid) (#1) was confirmed.

[Formula 1]

Then, a single component was separated from the CG-1 fraction, and as a result of LC-MS measurement, ion peaks of m/z 743, 611, 465, and 303 were confirmed. It has m/z 743 as a parent molecule and has m/z 611 and m/z 303, indicating that it is a qercetin glycoside. confirmed that there is. And the ion pattern from which rhamnose (m/z 146) was removed was confirmed from m/z 611. Based on the above results, it was identified as Quercetin [3-O-beta-(2(G)-O-beta-xylopyranosyl-6(G)-O-alpha-rhamnopyranosyl)glucopyranoside](#2) having the structure of Formula 2 .

[M+Na] ⁺ 765; [M+H] ⁺ 743; [M+H-xyl] ⁺ 611; [M+H-Rham-xyl] ⁺ 465; [quercetin aglycone] ⁺ 303;

[Formula 2]

Then, a single component was isolated from the CG-2 fraction, and it was first confirmed by comparative analysis with a rutin standard purchased from Sigma-aldrich using high-performance liquid chromatography under the conditions shown in Table 1 below. And as a result of LC-MS measurement, m/z 611 as a parent molecule and m/z 303 were confirmed to be Quercetin glycosides, and rhamnose (m/z 146) and glucose (m/z 162) were removed. The same ion pattern was confirmed. Based on the above results, Rutin [2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-[α-L-rhamnopyranosyl-(1→6)-β-D-glucopyanosyloxy]- 4H-chromen-4-one] (#3).

[M+Na] ⁺ 633; [M+H] ⁺ 611; [M+H-Rham] ⁺ 465; [quercetin aglycone] ⁺ 303;

[Table 1]

[Formula 3]

Example 2. Analysis of ability to inhibit expression of Rab27A and MyosinVa, which are target factors of melanin migration

In order to confirm the expression level of Rab27A, a major marker of melanin migration in melanocytes, which are mouse melanocytes, an experiment was performed as follows with reference to the method described in the literature (Hong JY et al. (2011) J Nat Prod 74). :2012-8).

RPMI medium containing 10% FBS was injected at a concentration of 1× ^{10 6} in a 100 mm culture dish, incubated for 24 hours at 37° C. and 5% CO ₂ conditions, and washed twice with phosphate buffered saline (PBS). After recovering the adherent cells and washing twice with PBS, boiling 2x sample loading buffer (250 mM Tris-HCl (pH 6.8), 4% SDS, 10% glycerol, 0.006% bromophenol blue, 2% β-mercaptoethanol, 50 mM sodium fluoride and 5 mM sodium orthovanadate) were added to disrupt the cells and maintained at 100° C. for 5 minutes. After cooling, it was stored at 20°C, and immediately before use, it was dissolved at 37°C and used for protein quantification and electrophoresis. 80 μg of protein was electrophoresed at 138 V for 2 hours using 5 ~ 12 % SDS-polyacrylamide gel (#456-1036, Bio-rad, Hercules, CA). The separated protein was transferred to a PVDF membrane (membrane, ISEQ15150, Millipore, Bedford, MA) at 100 V for 1 hour, washed twice with PBST, and a blocking buffer (5% non-fat dry milk in PBS containing 0.1% Tween-20) (PBST)) and incubated on a shaker at room temperature for 1 hour. Then, after washing 3 times for 5 minutes with PBST, the single antibody was diluted with PBST with 3% non-fat dry milk and reacted with a membrane at 4°C for 18 hours. The membrane [Membrane, ISEQ 1S150, Millipore, Bedford, MA) was washed 3 times for 5 minutes with PBST, and then the HRP (horseradish peroxidase)-conjugated secondary antibody was used with PBST and 3% skim milk powder at a ratio of 1:1,000 to 1:2,000. It was diluted and reacted with a membrane at room temperature for 3 to 4 hours. After washing 3 times for 5 minutes with PBST, it was treated with a chemiluminescent reagent (LabFrontier, Suwon, Korea). The protein expression levels of Rab27A and β-actin were confirmed in LAS-3000 (Fuji Film Corp., Japan). The results are shown in FIG. 2 . It was confirmed that the expression of Rab27A protein was inhibited in a concentration-dependent manner by caffeoylmalic acid isolated and identified from soybeans.

In addition, the melan-A cell line was plated with 2 x 10 ⁴ cells/ml in a confocal dish, proliferated for 24 hours, washed with PBS to completely remove FBS from the cells, fixed with 4% paraformaldehyde for 15 minutes, and then 0.1% Triton X Permeabilization was performed for 5 minutes using -100. Then, blocking was performed at room temperature for 30 minutes using 1% BSA, and then incubated in the primary antibody solution at 4° C. overnight. After incubating the fluorescence-conjugated secondary antibody at room temperature for 2 hours, counterstaining with DAPI (0.5 μg/ml) was performed, and then images were taken using a confocal microscope. The results are shown in FIG. 3 . It was confirmed that MyosinVa protein expression was decreased in a concentration-dependent manner according to the treatment with 20, 40, and 60 μM of caffeoyl malic acid isolated and identified from the soybean extract.

Example 3. Evaluation of skin whitening efficacy

Additionally, tyrosinase inhibitory efficacy in animal cell lines was measured by measuring the melanogenesis inhibitory activity of melan-A cells, a melanocyte cell line of mice treated with green bean extract. Melanin quantification was used by modifying the method of Hosoi et al. (Hosoi J et al., Cancer Res., 45(4), pp1474-1478, 1985).

Melan-a cells, which are melanocytes from mice, were dispensed in a 6-well plate at 3×10 ⁴ cells/well and cultured for 48 hours to attach the cells, Caffeoylmalic acid was treated to a final concentration of 20 μM and cultured for 3 to 5 days. The cultured cells were washed twice with phosphate buffer (DPBS; Dulbecco's Phosphated Buffered Saline) that does not contain calcium chloride (CaCl ₂ ), magnesium chloride (MgCl ₂ )) and treated with trypsin (trypsin, Gibco, USA) to separate the cells. Equal number of cells were collected for each group. The cell precipitate was washed with a phosphate buffer (DPBS; Dulbecco's Phosphated Buffered Saline) that does not contain calcium chloride (CaCl ₂ )) and magnesium chloride (MgCl ₂ ), and 100 μl of 10% dimethyl sulfoxide (DMSO) was added to 1 normal After being suspended in a sodium hydroxide (NaOH) solution of concentration (N), treated at 60 ° C. for 30 minutes, absorbance was measured at 475 nm, and the amount of melanin was converted from a standard curve made of synthetic melanin to show the inhibition rate as shown in FIG. Similarly, when 20 μM of caffeoylmalic acid was added, it was statistically significant (*P < 0.05) and inhibited 24.4% of melanin synthesis.

Therefore, it was found that caffeoyl malic acid isolated and identified from bean leaf extract has a very high potential for use as a cosmetic composition with whitening effect by inhibiting melanosome biosynthesis as well as melanosome mobility.

Example 4. Evaluation of photoaging inhibitory efficacy (Inflammaging target study)

배양접시에 2.2×10⁵ cells/ml plating 하여 24시간 동안 세포를 증식시킨 후 cell confluency가 90% 정도 도달했을때 PBS로 3번 세척하여 세포에 있는 FBS을 완전히 제거하였다. 이후 작두콩잎 추출물로부터 분리동정된 #1 (caffeoylmalic acid), #2 (Quercetin 3-O-beta-(2(G)-O-beta-xylopyranosyl- 6(G)-O-alpha-rhamnopyranosyl)glucopyranoside, #3 (rutin)가 각각 20 μM가 함유된 배지 (0.5 % FBS, 1 % AA in colorless DMEM)를 원래 배지량의 1/4 용량만 넣어 15 mJ/cm² UVB 조사후 남은 배지를 넣어 37℃ 5% CO₂ incubator에서 6시간 처리하였다. 그 결과는 도 5에 기재되어 있다. 대표적인 항염 효소계인 COX-2 단백질 발현량이 대조구에 비해 UV 조사시 현저하게 증가하였으며 20μg/ml 농도에서 작두콩잎 추출물로부터 분리 동정된 #1, 2, 및 3 물질로부터 광노화 억제효능을 분석한 결과 COX-2 발현이 현저하게 억제되는 것을 확인하였다. In order to evaluate the photoaging inhibitory effect of the green soybean extract of the present application, a HaCaT cell line, a human keratinocyte, was used for 30

After plating 2.2×10 ⁵ cells/ml on a culture dish, the cells were proliferated for 24 hours, and when the cell confluency reached about 90%, the cells were washed 3 times with PBS to completely remove FBS from the cells. #1 (caffeoylmalic acid), #2 (Quercetin 3-O-beta-(2(G)-O-beta-xylopyranosyl-6(G)-O-alpha-rhamnopyranosyl)glucopyranoside, Add only 1/4 volume of the original medium (0.5% FBS, 1% AA in colorless DMEM) containing 20 μM of #3 (rutin), 15 mJ/cm ² Add the remaining medium after UVB irradiation at 37℃ 5 % CO ₂ was treated for 6 hours in an incubator The results are shown in Fig. The expression level of COX-2 protein, a representative anti-inflammatory enzyme system, was significantly increased upon UV irradiation compared to the control, and was isolated from the green bean leaf extract at a concentration of 20 μg/ml As a result of analyzing the photoaging inhibitory effect from the identified #1, 2, and 3 substances, it was confirmed that COX-2 expression was remarkably inhibited.

Hereinafter, a cream composition, a massage cream composition, a lotion composition, a skin lotion composition, an essence composition, a pack composition, and a cleansing foam composition are exemplified as formulation examples of the present invention, but the formulation including the cosmetic composition of the present invention is limited thereto not.

Formulation Example 1. Cream composition

After heating and mixing the oil phase and the aqueous phase at 75° C., respectively, with the composition and content (% by weight) shown in the table below, the mixture is cooled to room temperature.

Formulation Example 2. Massage Cream Composition

The oil phase and the aqueous phase are each heated to 75° C. to dissolve and mix with the composition and content (% by weight) shown in the table below, and then cooled to room temperature.

Formulation Example 3. Lotion composition

After emulsifying the oil phase and the aqueous phase at 75° C., respectively, with the composition and content (% by weight) shown in the table below, the mixture is cooled to room temperature.

Formulation Example 4. Skin lotion composition

The aqueous phase and the ethanol phase are prepared and mixed with the composition and content (% by weight) shown in the table below, and then filtered.

Formulation Example 5. Essence composition

The aqueous phase and the ethanol phase are prepared and mixed with the composition and content (% by weight) shown in the table below, and then filtered.

Formulation Example 6. Pack composition

Dispersing and dissolving the aqueous phase and the ethanol phase, respectively, with the composition and content (wt%) described in the table below, mixing, and then cooling to room temperature.

Formulation Example 7. Cleansing Foam Composition

After dispersing and dissolving each of the aqueous phase and the oil phase with the composition and content (% by weight) shown in the table below, mixing and saponification, the mixture is cooled to room temperature.

In addition, as formulation examples of the cosmetic food of the present invention, chewing gum, candy, biscuit, ice cream, sausage, chocolate, general beverage and whitening probiotic lactic acid bacteria beverage formulation are exemplified, but the formulation containing the cosmetic food composition of the present invention is limited thereto not.

Preparation Example 1. Preparation of Chewing Gum (A)

Chewing gum was prepared in the usual manner with the composition and content shown in the table below.

Preparation Example 2. Preparation of Chewing Gum (B)

Chewing gum was prepared in the usual manner with the composition and content shown in the table below.

Preparation Example 3. Preparation of Candy (A)

Candy was prepared according to the composition and content shown in the table below and the conventional method.

Preparation Example 4. Preparation of Candy (B)

Candy was prepared according to the composition and content shown in the table below and the conventional method.

Preparation 5. Preparation of biscuits

Biscuits were prepared according to the composition and content in the table below and the conventional method.

Preparation Example 6. Preparation of ice cream

Ice cream was prepared in a conventional manner with the composition and content shown in the table below.

Preparation Example 7. Preparation of Sausage

Sausages were prepared in the usual manner with the composition and content of the table below.

Production Example 8. Production of chocolate

Chocolate was prepared in a conventional manner with the composition and content of the table below.

Preparation 9. Preparation of health food

Health food was prepared in the usual manner with the composition and content of the table below.

The composition ratio of the vitamin and mineral mixture is a composition that is relatively suitable for health food in a preferred embodiment, but the mixing ratio may be arbitrarily modified. , to prepare granules, and can be used in the manufacture of health food compositions according to a conventional method.

Preparation Example 10. Preparation of health drinks

A health drink was prepared in the usual manner with the composition and content of the table below.

After mixing the above ingredients according to a conventional health drink manufacturing method, stirring and heating at 85° C. for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2L container, sealed and sterilized, and then refrigerated. used in the manufacture of health beverage compositions of

Although the composition ratio is prepared by mixing ingredients suitable for relatively favorite beverages in a preferred embodiment, the mixing ratio may be arbitrarily modified according to regional and national preferences such as demand class, demanding country, and use.

Claims (10)

A cosmetic composition for skin whitening and photoaging inhibition comprising caffeoyl malic acid.
The method of claim 1,
The composition comprises quercetin 3-O-beta-(2(G)-O-beta-xylopyranosyl-6(G)-O-alpha-rhamnopyranosyl)glucopyranoside) or rutin (2-(3,4-dihydroxyphenyl)-5, 7-dihydroxy-3-[α-L-rhamnopyranosyl-(1→6)-β-D-glucopyanosyloxy]-4H-chromen-4-one) which further comprises one or more of, skin whitening and photoaging inhibition cosmetic composition for use.
The method of claim 1,
The skin whitening inhibits the movement of melanin generated in melanin forming cells constituting the epidermal cells of the skin to the keratinocytes; And by inhibiting melanin production in melanocytes, a cosmetic composition for skin whitening and photoaging inhibition.
4. The method of claim 3,
The inhibition of the migration of melanin into keratinocytes is by suppression of the expression of Rab27A and MyosinVa involved therein, a cosmetic composition for skin whitening and photoaging inhibition.
3. The method according to claim 1 or 2,
The photoaging inhibition is by inhibition of COX-2 (cyclooxygenase-2) expression, a cosmetic composition for skin whitening and inhibition of photoaging.
A functional food composition for skin whitening and photoaging improvement, comprising caffeoyl malic acid.
7. The method of claim 6,
The composition comprises quercetin 3-O-beta-(2(G)-O-beta-xylopyranosyl-6(G)-O-alpha-rhamnopyranosyl)glucopyranoside) or rutin (2-(3,4-dihydroxyphenyl)-5, 7-dihydroxy-3-[α-L-rhamnopyranosyl-(1→6)-β-D-glucopyanosyloxy]-4H-chromen-4-one) to further include one or more of, skin whitening and photoaging improvement functional food composition for use.
7. The method of claim 6,
The skin whitening inhibits the movement of melanin produced in melanin forming cells constituting epidermal cells to keratinocytes; And / or by inhibiting melanin production in melanocytes, skin whitening and photoaging improvement functional food composition.
9. The method of claim 8,
Inhibition of the movement of melanin into keratinocytes is by suppression of the expression of Rab27A and MyosinVa involved therein, a functional food composition for skin whitening and photoaging improvement.
7. The method of claim 6,
The photoaging inhibition is by inhibition of COX-2 (cyclooxygenase-2) expression, skin whitening and functional food composition for improving photoaging.

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