KR20210011255A - Compostions for whitening comprising compounds or extracts of Osmanthus fragrans - Google Patents

Abstract

The present invention relates to a cosmetic composition for skin whitening including an extract of Osmanthus fragrans, a fraction thereof, and at least one substance selected from the group consisting of 3alpha,24-dihydroxy-urs-12-en-28-oic acid, pomolic acid, maslinic acid, corosolic acid and cosmetically acceptable salts of the compounds. The composition shows an excellent effect of inhibiting melanin formation and tyrosinase-inhibiting activity, is a material safe to the human body, and is useful for skin whitening.

Description

Composition for whitening comprising extracts of Geummokseo and compounds isolated therefrom {Compostions for whitening comprising compounds or extracts of Osmanthus fragrans}

The present invention relates to a composition for skin whitening comprising an extract of Geummokseo, a fraction thereof and/or a compound isolated therefrom.

Human skin color is determined by the concentration and distribution of melanin inside the skin. This concentration and distribution of melanin is affected by environmental or physiological conditions such as solar ultraviolet rays, fatigue, and stress in addition to genetic factors. Melanin pigment produced by melanocytes of human skin is a phenolic polymer material having a complex form of black pigment and protein, and plays an important role in blocking skin damage caused by ultraviolet rays. It has been reported that the action of tyrosinase present in melanocytes is the most important for melanin biosynthesis. Tyrosinase plays a key role in the skin blackening process by converting tyrosine, a type of amino acid, into dopa and dopaquinone, intermediate products of melanin polymer production. However, although the pathway by which melanin is made is known, the mechanism that induces melanin synthesis, a previous step in which tyrosinase acts, is still not elucidated in detail.

When such melanin synthesis occurs excessively in the skin, it darkens the skin tone and causes spots and freckles. Therefore, by inhibiting the synthesis of melanin pigment in the skin, not only can the skin tone be brightened and skin whitening can be realized, but also skin hyperpigmentation such as spots and freckles caused by ultraviolet rays, hormones and genetic causes can be improved. have.

Therefore, conventionally, substances having inhibitory activity against tyrosinase such as hydroquinone, ascorbic acid, kojic acid, and glutathione have been used to reduce skin whitening or skin hyperpigmentation. Improved. However, although hydroquinone exerts a predetermined whitening effect, it has a problem that the amount of ascorbic acid should be limited to a very small amount due to severe skin irritation, and ascorbic acid is easily oxidized, causing problems such as discoloration and discoloration of the cosmetic composition. Kojic acid is unstable in the solution, which complicates the manufacturing process of cosmetics. In addition, thiol-based compounds such as glutathione and cysteine not only have a peculiar unpleasant odor, but also have problems in transdermal absorption, and their glycosides and derivatives are also highly polar, making it difficult to use as a cosmetic ingredient. In addition, in the case of vitamin C, there is a problem that it is easily oxidized in an aqueous solution state and does not produce a lasting effect. Accordingly, recently, compositions for skin whitening including natural herbal extracts have been developed, but there is a problem in the formulation because most of them are colored, and there is a problem that the same effect cannot be expected during product manufacturing because the active ingredient has not been identified.

On the other hand, Geummokseo ( Osmanthus fragrans var. aurantiacus ) is an evergreen small arboreous tree of the dicotyledonous gentian ash family. When it grows, the height is 3 ~ 4m, the leaves are opposite, long oval-shaped, broad lanceolate, densely attached, finely serrated or flat on the edge of the leaf, the surface is dark green, and the back side is light green. In addition, the flowers of Geummokseo bloom from the end of September to the beginning of October, and have a very good scent in yellow.

Looking at what has been studied about the Geummokseo, Korean Patent Publication No. 10-2019-0058168 discloses an antioxidant or anti-inflammatory composition containing an extract of Geummokseo, and Korean Patent Laid-Open No. 10-2016-0108685 is the same as Geummokseo. Disclosed is a fragrance composition that reproduces the fragrance of Eunmokseo belonging to a heterogeneous species. As such, there has not been much research on Geummokseo.

Accordingly, the present inventors discovered that the extract of Geummokseo, its fractions, and the compounds isolated therefrom were particularly excellent in whitening effect, high stability, and no skin side effects, as a result of which the present invention was completed. .

Therefore, one object of the present invention is to provide a composition for whitening comprising a Geummokseo extract or a fraction thereof.

Another object of the present invention is 3 alpha,24-dihydroxy-urs-12-den-28-oic acid (3α,24-dihydroxy-urs-12-en-28-oic acid), pomolic acid acid), maslinic acid, corsolic acid, and cosmetically acceptable salts of these compounds to provide a composition for skin whitening comprising one or more compounds selected from the group consisting of.

Another object of the present invention is to provide the composition as a cosmetic composition for skin whitening.

Another object of the present invention is to provide a method for reducing the concentration of melanin in the skin of an individual by administering the composition to an individual for skin whitening.

In one aspect, the present invention relates to a cosmetic composition for skin whitening comprising an extract or a fraction thereof, Geummokseo ( Osmanthus fragrans var. aurantiacus ).

In the present invention, "Geummokseo" refers to a plant of the genus of the ash family, including roots, stems, leaves, or fruits including natural, hybrids or varieties thereof.

In the present invention, the extract and fraction refer to a substance having an activity of inhibiting tyrosinase activity and melanin production isolated from natural products. In addition, in the present invention, extracts and fractions thereof are used in the sense to include not only extracts or fractions of a plant solvent, but also purified products, dry powders thereof, or all forms formulated using the same.

The purified product is a process of removing suspended solid particles from an extract or fraction, and the particles may be filtered using cotton, nylon, or the like, or an ultrafiltration method, a cryofiltration method, a centrifugation method, etc. may be used, but is not limited thereto.

In addition, a separation process by various chromatography (chromatography according to size, charge, hydrophobicity or affinity) may be additionally included.

The extract, fraction, or purified product may be used as it is in a liquid form, or it may be used after concentration and/or drying. The concentration and/or drying method is not limited thereto, but includes methods such as freeze drying, vacuum drying, hot air drying, spray drying, vacuum drying, foam drying, high frequency drying, infrared drying, etc.

The Geummokseo extract may be extracted using water, an organic solvent, or a mixed solvent thereof, and preferably, may be extracted using an organic solvent. The extracted liquid can be used in a liquid form or concentrated and/or dried.

For extraction using an organic solvent, methanol, ethanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or an organic solvent that is a mixed solvent thereof is used, and the active ingredient of the extract is not destroyed or can be extracted by heating at room temperature or under a minimized condition. Depending on the organic solvent to be extracted, the degree of extraction and the degree of loss of the active ingredient of the extract may be different, so select and use an appropriate organic solvent.

In one specific embodiment, the extract of Geummokseo of the present invention is 3 to 20 times, preferably 3 to 15 times of water, lower alcohol of C1 to C4, or these A mixed solvent of, preferably an ethanol or methanol solvent; 10 to 40°C, preferably 15 to 37°C, extraction temperature; 1 hour to 15 days, preferably 1 hour to 1 day; 1 to 5 times, preferably 1 to 3 times; The extract obtained by using a cold needle, ultrasonic extraction, reflux cooling extraction, preferably a cold needle extraction method was obtained by filtration and concentration under reduced pressure.

The fraction of the Geummokseo extract may be fractionated from the extract using an organic solvent or a mixed solvent thereof, and the fraction may be used in a liquid form or concentrated and/or dried.

The solvent for the fraction is methanol, ethanol, propanol, isopropanol, normal butanol, isobutanol, pentanol, hexanol, ethylene, acetone, normal hexane, isohexane, cyclohexane, ether, chloroform, ethyl acetate, butyl acetate, dichloro Methane, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, and water may be used alone or as a mixture of two or more, but are not limited thereto. Preferably, they are normal hexanol, ethyl acetate, normal butanol and water.

In one specific implementation, a Geummokseo leaf extract was prepared by adding ethanol to Geummokseo leaves, and then fractionated sequentially using normal hexane, ethyl acetate, normal butanol, and water.

The Geummokseo extract and its fractions obtained by the above manufacturing method inhibit the activity of tyrosinase and effectively inhibit melanin production, so that the skin whitening effect is excellent.

In addition, since the Geummokseo extract and its fractions have almost no toxicity and side effects, they can be safely used in the composition for skin whitening of the present invention.

Geummokseo extract and/or fractions thereof obtained by the above manufacturing method may be included in 0.0001 to 15% by weight, preferably 0.001 to 15% by weight, more preferably 0.001 to 5% by weight, based on the total weight of the composition of the present invention. have. If it is less than 0.0001% by weight, the whitening effect is too weak, and if it exceeds 15% by weight, there is a problem in that the increase in the effect due to the increase in the content is insufficient and the stability of the formulation is not secured.

In another aspect, the present invention provides 3 alpha,24-dihydroxy-urs-12-den-28-oic acid (3α,24-dihydroxy-urs-12-en-28-oic acid), formalic acid ( Provides a composition for skin whitening comprising one or more compounds selected from the group consisting of pomolic acid), maslinic acid, corsolic acid, and pharmaceutical or cosmetically acceptable salts of these compounds. .

The 3-alpha,24-dihydroxy-urs-12-den-28-oic acid (3α,24-dihydroxy-urs-12-en-28-oic acid) is a compound having the structure of Formula 1 below, It is called "Compound 1".

[Formula 1]

The pomolic acid is a compound having a structure represented by the following formula (2), and is hereinafter referred to as "compound 2".

[Formula 2]

Maslinic acid (maslinic acid) is a compound having the structure of the following formula (3), hereinafter referred to as "compound 3".

[Formula 3]

Corsolic acid (corsolic acid) is a compound having the structure of the following formula (4), hereinafter referred to as "compound 4".

[Formula 4]

The compounds 1 to 4 are prepared according to known manufacturing methods for them, for example, extracted and purified from Osmanthus fragrans var. aurantiacus , or microbial culture such as Saccharomyces cerevisiae It may be a product obtained through or chemically synthesized. Each of the compounds 1 to 4 may have a molecular formula of C ₃₀ H ₄₈ O ₄ .

The compounds 1 to 4 of the present invention have a concentration-dependent increase in the inhibitory effect of intracellular melanin production, and in particular, compounds 1 and 3 have a significantly higher melanin production inhibitory effect compared to Arbutin, which is used as an ingredient for whitening cosmetics. Indicated. Therefore, the compounds 1 to 4 of the present invention are excellent in whitening effect due to inhibition of melanin production.

The compounds 1 to 4 of the present invention may be used in the form of a cosmetically acceptable salt, and as such a salt, an acid addition salt formed by a cosmetically acceptable free acid or a metal salt formed by a base have. Inorganic acids and organic acids may be used as the free acid, hydrochloric acid, sulfuric acid, bromic acid, sulfurous acid or phosphoric acid may be used as the inorganic acid, and citric acid, acetic acid, maleic acid, humaic acid, gluconic acid, methanesulfonic acid, etc. may be used as the organic acid. It may be used, but is not limited thereto. The metal salt includes an alkali metal salt or an alkaline earth metal salt, and sodium, potassium or calcium salts are useful.

In the composition for skin whitening of the present invention, the compounds 1 to 4 or salts of these compounds are preferably 0.0001 to 15% by weight based on the total composition. If it is less than 0.0001% by weight, the whitening effect is too weak, and if it exceeds 15% by weight, there is a problem in that the increase in the effect due to the increase in the content is insufficient and the stability of the formulation is not secured. More preferably, the compounds 1 to 4 or salts of these compounds are contained in an amount of 0.001 to 5% by weight based on the total composition.

The composition of the present invention may be a cosmetic composition for skin whitening.

The cosmetic composition is an active ingredient, Geummokseo extract, a fraction thereof, and 3α,24-dihydroxy-urs-12-den-28-oic acid (3α,24-dihydroxy-urs-12-en-28-oic acid). ), pomolic acid, maslinic acid, corsolic acid, and one or two or more substances selected from cosmetically acceptable salts of these compounds, as well as ingredients commonly used in cosmetic compositions And, for example, antioxidants, stabilizers, solubilizers, vitamins, pigments, and conventional auxiliaries and/or carriers such as fragrances. In addition, the cosmetic composition may further include a skin absorption promoting material in order to enhance the skin whitening effect.

The cosmetic composition of the present invention may be prepared in any formulation commonly 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. may be formulated, but is not limited thereto. More specifically, it may be prepared in the form of a flexible lotion, nutritional lotion, nutritional cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder.

When the formulation of the present invention is a paste, cream, or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide are used as carrier components. Can be.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additionally chlorofluorohydrocarbon, propane / May contain propellants such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

When the formulation of the present invention is a suspension, liquid diluents such as water, ethanol or propylene glycol as carrier components, ethoxylated isostearyl alcohol, suspending agents such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, crystallites Sex cellulose, aluminum metahydroxide, bentonite, agar or tragicanth, and the like can be used.

When the formulation of the present invention is a surfactant containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters may be used.

In the cosmetic composition of the present invention, Geummokseo extract, its fraction, and 3α,24-dihydroxy-urs-12-den-28-oic acid (3α,24-dihydroxy-urs-12-en-28-oic acid) ), pomolic acid, maslinic acid, corsolic acid, and the amount of one or two or more substances selected from cosmetically acceptable salts of these compounds is 0.0001 to 15% by weight based on the total composition , Preferably 0.001 to 15% by weight, more preferably 0.001 to 5% by weight may be included.

In another aspect, the present invention provides a method for whitening skin by reducing the concentration of melanin in the skin of the individual by administering the composition to an individual for skin whitening.

The individual is a mammal, including humans, which can achieve the purpose of skin whitening by administration of the composition of the present invention, for example, monkeys, cows, horses, pigs, sheep, dogs, cats, rats, mice, It means chimpanzees, etc.

The administration means introducing a predetermined substance to the individual by any suitable method, and the administration route of the composition of the present invention may be administered orally or parenterally through any general route as long as it can reach the target tissue. In addition, the compositions of the present invention can be administered by any device capable of moving the active substance to a target subject, eg, a cell.

A suitable dosage of the composition of the present invention varies depending on the condition and weight of the individual, the severity of the disease, the form of the drug, and the time, but may be appropriately selected by those skilled in the art. The daily dosage of the composition is 1.0 ㎖ to 3.0 ㎖ based on an adult, and may be administered by applying once to 5 times a day as needed.

On the other hand, in a specific embodiment of the present invention, Geummokseo extract, a fraction thereof, and 3alpha,24-dihydroxy-urs-12-den-28-oic acid (3α,24-dihydroxy-urs-12-en-28- oic acid), pomolic acid, maslinic acid, corsolic acid, and cosmetically acceptable salts of these compounds, as a result of cytotoxicity tests, were found to be natural substances and harmless to humans. . Therefore, the extract of the present invention, a fraction thereof, and 3α,24-dihydroxy-urs-12-den-28-oic acid (3α,24-dihydroxy-urs-12-en-28-oic acid), Pomolic acid, maslinic acid, corsolic acid, and cosmetically acceptable salts of these compounds have little toxicity and side effects, so they can be safely used even when used for a long time. It can be safely applied to such cosmetic compositions.

According to the present invention, Geummokseo extract, a fraction thereof, and 3α,24-dihydroxy-urs-12-den-28-oic acid (3α,24-dihydroxy-urs-12-en-28-oic acid), formol Pomolic acid, maslinic acid, corsolic acid, and cosmetically acceptable salts of these compounds have an inhibitory effect on melanogenesis. In addition, it was found to be a safe material for human use as it does not generate any toxicity or irritation in the human safety test. Therefore, the material of the present invention can be usefully used in cosmetics and medicines for skin whitening.

1 is a diagram showing the cell viability (%) according to the concentration of the Geummokseo leaf extract and fraction according to an embodiment of the present invention.
Figure 2 is 3 alpha, 24-dihydroxy-urs-12-den-28-oic acid (3α,24-dihydroxy-urs-12-en-28-oic acid) according to an embodiment of the present invention, formol It is a figure showing the cell viability (%) according to the concentration of the compounds of pomolic acid, maslinic acid, and corsolic acid.
3 is a view showing the melanin content (%) according to the concentration of the extract and fractions of Geummokseo leaf according to an embodiment of the present invention.
Figure 4 is a 3-alpha, 24-dihydroxy-urs-12-den-28-oic acid (3α,24-dihydroxy-urs-12-en-28-oic acid) according to an embodiment of the present invention, formol The figure shows the melanin content (%) according to the concentration of the compounds of pomolic acid, maslinic acid, and corsolic acid.

The present invention will be described in more detail through the following examples. However, the present invention is not limited thereto.

Example 1: Preparation of extract of Geummokseo

Gummokseo leaves were collected at Seoul National University's Southern Academic Forest (located in Baegunsan, Gwangyang-si, Jeollanam-do), added 100% ethanol to 1 g of the collected sample, extracted with ultrasonic waves in a 37℃ water bath for 2 hours, concentrated under reduced pressure, and diluted with distilled water to use. I did. The diluted sample was used for later experiments by blocking the light and keeping it frozen.

Example 2: Isolation and identification of compounds

The Geummokseo leaf extract prepared in Example 1 was sequentially fractionated in order of n-Hexane, Etylacetate, n-Butanol, and Water, concentrated under reduced pressure, and diluted with distilled water to use. The diluted sample was used for later experiments while keeping the light blocked and frozen.

After filtering the fraction, the compound was separated using semi-HPLC, and the structure was identified using NMR. As a result, it was found that it is a compound represented by the following Chemical Formulas 1 to 4.

Compound 1: 3α,24-dihydroxy-urs-12-den-28-oic acid (3α,24-dihydroxy-urs-12-en-28-oic acid)

In order to identify the structure of Compound 1, ¹ H-NMR spectrum and ¹³ C-NMR spectrum were measured.

^{In 1} H-NMR spectrum, 7 methyl group proton signals ( δ _H 1.16, 3H, s, H-27; 1.07, 3H, s, H-26; 1.04, 3H, s, H-23; 0.96, 3H, d , J=6.4Hz, H-29; 0.95, 3H, s, H-25; 0.92, 3H, d, J=6.4, H-30). H-29 and H-30 were expected to be ursane triterpenoids as they appeared as doublet methyl protons. Since H-24 was shifted to a high magnetic field to 4.19 (1H, d, 10.0Hz, H-24α) and 3.72 (1H, d, 10.4Hz, H-24β), it was predicted that the OH group was substituted. ^{In the 13} C-NMR spectrum, it is about 37 ppm (C-1) in the case of 3α-OH, compared to 39 ppm (C-1) in the case of 3β-OH carbon, and 3β from compound 1 to ( δ _C 38.9, C-1). Identified with -OH. In addition, urs-12-ene carbon signal ( δ _C 139.2, C-13), carbonyl carbon signal ( δ _C 179.9, C-28) were identified, and six methyl carbon signals ( δ _C 23.9, C-27; 23.6). , C-11; 21.4, C-30; 18.5, C-6; 17.5, C-26; 17.5, C-29; 16.1, C-25; 13.1, C-23), 12 methylene signals ( δ _C 125.6 , C-12; 73.4, C-3; 67.8, C-24; 38.9, C-1; 37.4, C-22; 33.2, C-7; 31.0, C-21; 28.7, C-15; 27.7, C -2; 24.9, C-16; 23.6, C-11; 18.5, C-6), 5 methine signals ( δ _C 53.5, C-18; 48.5, C-5; 48.0, C-9; 39.4, C -19; 39.4, C-20), 7 quaternary carbon signals ( δ _C 179.9, C-28; 139.2, C-13; 48.0, C-17; 42.9, C-4; 42.5, C-14; 39.9, C-8; 37.1, C-10) was confirmed. In addition, C-24 was confirmed at 67.8 ppm in which the OH group was substituted and the high magnetic field was shifted. The position of each substituent was confirmed through 2D NMR spectrum. It was confirmed that the molecular formula of Compound 1 is C ₃₀ H ₄₈ O ₄ .

[Formula 1]

Compound 2: pomolic acid

In order to identify the structure of Compound 2, ¹ H-NMR spectrum and ¹³ C-NMR spectrum were measured.

^{In 1} H-NMR spectrum, 7 methyl group proton signals ( δ _H 1.73, 3H, s, H-29; 1.45, 3H, s, H-27; 1.23, 3H, s, H-25; 1.12, 3H, s , H-23; 1.11, 3H, d, J=6.4Hz, H-30; 1.02, 3H, s, H-24; 0.91, 3H, s, H-26). It was expected that H-29 was a singlet methyl proton with an OH group substituted on C-19, and H-30 was expected to be a ursane triterpenoid as it appeared as a doublet methyl proton. ^{In the 13} C-NMR spectrum, when 3α-OH is about 37 ppm (C-1), about 76 ppm (C-3), about 22 ppm (C-24), 3β-OH carbon is 39 ppm (C -1), about 79 ppm (C-3), about 16 ppm (C-24) in compound 2 as ( δ _C 39.0, C-1; 78.2 C-3; 16.8 C-24) as 3β-OH I did. In addition, urs-12-ene carbon signal ( δ _C 140.0, C-13) and carbonyl carbon signal ( δ _C 180.7, C-28) were identified, and seven methyl carbon signals ( δ _C 28.8, C-23; 26.9). , C-29; 24.7, C-27; 17.2, C-26; 16.8, C-24; 16.5, C-30; 15.6, C-25), 9 methylene signals ( δ _C 39.0, C-1; 38.6 , C-22; 33.6, C-7; 29.3, C-15; 28.1, C-2; 27.1, C-21; 26.4, C-16; 24.0, C-11; 18.9, C-6), 6 methine signal ( δ _C 128.0, C-12; 78.2, C-3; 55.9, C-5; 54.6, C-18; 47.8, C-9; 42.4, C-20), 8 quaternary carbon signals ( δ _C 180.7, C-28; 140.0, C-13; 72.7, C-19; 48.3, C-17; 42.1, C-14; 40.4, C-8; 39.4, C-4; 37.4, C-10) I did. In addition, C-19 was confirmed at 72.7 ppm where the OH group was substituted and the high magnetic field was shifted. The position of each substituent was confirmed through 2D NMR spectrum. It was confirmed that the molecular formula of Compound 2 is C ₃₀ H ₄₈ O ₄ .

[Formula 2]

Compound 3: maslinic acid

In order to identify the structure of Compound 3, ¹ H-NMR spectrum and ¹³ C-NMR spectrum were measured.

^{In 1} H-NMR spectrum, the hydroxyl groups present in C-2 and C-3 are ( δ _H 4.10, 1H, td, J=10.0, 4,8 Hz, H-2; 3.39, 1H, d, J=9.6 Hz, H-3) can be seen to have C ₂ -α-OH, and C ₃ -β-OH. And 7 methyl group proton signals ( δ _H 1.27, 3H, s, H-23; 1.25, 3H, s, H-27; 1.07, 3H, s, H-24; 1.00, 3H, s, H-26; 0.98, 3H, s, H-30; 0.97, 3H, s, H-25; 0.93, 3H, s, H-29) were identified. Oleanane structure could be predicted as H-29 and H-30 came out as singlet. ^{In the 13} C-NMR spectrum, in the case of C ₂ -α-OH and C ₃ -α-OH, the chemical shifts of C-1 and C-2 are about 66.5 ppm and 78.9 ppm, respectively, whereas C ₂ -α- In the case of OH and C ₃ -β-OH, it is about 68.8 ppm and about 83.8 ppm, respectively, and it is slightly shifted in the low field, so compound 3 becomes ( δ _C 83.8, C-3; 68.6, C-2) It can be seen that it has 2-α-OH and 3-β-OH. In addition, olean-12-ene carbon signal ( δ _C 144.8, C-13) and carbonyl carbon signal ( δ _C 180.2, C-28) were identified, and seven methyl carbon signals ( δ _C 33.2, C-29; 29.3 , C-23; 26.1, C-27; 23.7, C-25; 17.7, C-26; 17.4, C-24; 16.8, C-30), 9 methylene signals ( δ _C 47.7, C-1; 42.0 , C-19; 34.2, C-21; 33.2, C-22; 33.2, C-7; 28.3, C-15; 24.0, C-16; 23.9, C-11; 18.8, C-6), 6 methine signal ( δ _C 122.4, C-12; 83.8, C-3; 68.6, C-2; 55.9, C-5; 48.1, C-9; 42.0, C-18), 8 quaternary carbon signals ( δ _C 180.2, C-28; 144.8, C-13; 46.6, C-17; 42.2, C-14; 39.8, C-8; 39.8, C-4; 38.5, C-20; 30.9, C-10) I did. The position of each substituent was confirmed through 2D NMR spectrum. It was confirmed that the molecular formula of compound 3 is C ₃₀ H ₄₈ O ₄ .

[Formula 3]

Compound 4: corsolic acid

In order to identify the structure of Compound 4, ¹ H-NMR spectrum and ¹³ C-NMR spectrum were measured.

¹ H-NMR spectrum shows 7 methyl group proton signals ( δ _H 1.27, 3H, s, H-23; 1.20, 3H, s, H-27; 1.07, 3H, s, H-24; 1.03, 3H, s , H-26; 0.97, 3H, d, J=6.0Hz, H-30; 0.97, 3H, s, H-25; 0.93, 3H, d, J=6.4, H-29). H-29 and H-30 were expected to be ursane triterpenoids as they appeared as doublet methyl protons. ^{In the 13} C-NMR spectrum, in the case of C ₂ -α-OH and C ₃ -α-OH, the chemical shifts of C-1 and C-2 are about 66.5 ppm and 78.9 ppm, respectively, whereas C ₂ -α- In the case of OH and C ₃ -β-OH, it is about 68.8 ppm and about 83.8 ppm, respectively, and it is slightly shifted in the low field, so compound 4 becomes ( δ _C 83.8, C-3; 68.6, C-2) It can be seen that it has 2-α-OH and 3-β-OH. In addition, urs-12-ene carbon signal ( δ _C 139.3, C-13) and carbonyl carbon signal ( δ _C 179.9, C-28) were identified, and seven methyl carbon signals ( δ _C 29.4, C-23; 23.9) , C-27; 21.4, C-29; 17.7, C-24; 17.5, C-26; 17.5, C-30; 17.0, C-25) methylene signal ( δ _C 48.0, C-1; 37.4, C- 22; 33.5, C-7; 31.1, C-21; 28.6, C-16; 24.9, C-15; 23.7, C-11; 18.8, C-6), methine signal ( ? _C 125.5, C-12; 83.8, C-3; 68.6, C-2; 55.9, C-5; 53.5, C-18; 48.1, C-9; 39.5, C-19; 39.4, C-20), quaternary carbon signal ( δ _C 179.9 , C-28; 139.3, C-13; 48.0, C-17; 42.5, C-14; 40.0, C-4; 39.8, C-8; 38.4, C-10) were identified. The position of each substituent was confirmed through 2D NMR spectrum. It was confirmed that the molecular formula of compound 4 is C ₃₀ H ₄₈ O ₄ .

[Formula 4]

Example 3: Safety evaluation for cytotoxicity of Geummokseo extract and compounds isolated therefrom

Add 2 x 10 ⁴ cells/well of mouse-derived melanoma cell B16F10 cells to a 96-well plate, add DMEM medium containing 10% FBS, and stabilize for 24 hours in 5% CO ₂ , 37°C incubator conditions, and After replacement, samples were added for each concentration, and then incubated for 72 hours.

The MTT solution was treated and the medium was removed after 4 hours in a dark room at 37°C, and formazan was dissolved using DMSO. The absorbance at 540 nm was measured, and the cell viability was expressed in% using Equation 1 below compared to the control group. The results are shown in FIGS. 1 to 2.

Equation 1

Cell viability% = 100 X (absorbance of treatment group / absorbance of control group)

1 is a result of measuring cytotoxicity by diluting and treating the leaf extract and fraction of Geummokseo at a concentration of 250 μg/mL. The extracts and fractions from leaves of Geummokseo were found to be non-cytotoxic at a concentration of 250 μg/mL.

2 is a result of measuring cytotoxicity after treatment by diluting the compounds of Formulas 1 to 4 isolated from Geummokseo to a concentration of 1 and 10 μM. Compounds of Formulas 1 to 4 isolated from Geummokseo were found to be free of cytotoxicity at concentrations of 1 and 10 μM.

Example 4: Melanogenesis inhibitory effect of Geummokseo extract and compounds isolated therefrom

Inhibitory effect of melanin production by Geummokseo extract, its fraction and the compound isolated therefrom was measured using mouse pigment cells (B16 melanoma cells, ATCC), and the inhibitory effect of melanin production by arbutin, which is known to inhibit melanogenesis. Compared with.

Add 2 x 10 ⁵ cells/well of mouse-derived melanoma cell B16F10 cells to a 24 well plate, add DMEM medium containing 10% FBS, and stabilize for 24 hours in 5% CO ₂ , 37°C incubator conditions, After replacement, samples were added for each concentration, and then incubated for 72 hours. After 72 hours incubation, 1N NaOH to which 10% DMSO was added was added and left at 80° C. for 1 hour to recover melanin in the cells. As for melanin, the amount of melanin produced in unit cells was measured at 490 nm using ELISA, and was expressed as a percentage of the control group. The results are shown in Figures 3 to 4.

3 is a result of measuring the melanin content after treatment by diluting the leaf extracts and fractions of Geummokseo at a concentration of 250 μg/mL. The melanin content of Geummokseo leaf extract decreased to 86.3% at the concentration of 250 μg/mL, and the melanin content of the fractions, especially at the concentration of 250 μg/mL of ethyl acetate, decreased to 73.4%. From these results, it was found that the extract and ethyl acetate fraction of the leaves of Geummokseo showed higher melanin inhibitory activity than the positive control, arbutin.

4 is a result of measuring the melanin content after treatment by diluting the compounds of Formulas 1 to 4 isolated from Geummokseo to a concentration of 1 and 10 μM. The compound of formula 1 is 68.0% and 58.0% at a concentration of 1 and 10 μM, the compound of formula 2 is 88.7% and 86.7%, the compound of formula 3 is 78.5% and 77.1%, the compound of formula 4 is 97.8%, The melanin content tended to decrease to 93.7% depending on the concentration. From this result, it was found that the compounds of Formulas 1 and 3 isolated from Geummokseo had higher melanin inhibitory activity than arbutin, a positive control.

Claims (4)

Geummokseo extract, a fraction of the Geummokseo extract, and Geummokseo extract, a fraction thereof, and 3alpha,24-dihydroxy-urs-12-den-28-oic acid (3α,24-dihydroxy-urs-12-en-28 -oic acid), pomolic acid, maslinic acid, corsolic acid, and skin whitening containing one or more substances selected from the group consisting of cosmetically acceptable salts of these compounds Cosmetic composition.
The cosmetic composition for skin whitening according to claim 1, wherein the Geummokseo extract is a leaf extract of Geummokseo.
The cosmetic composition for skin whitening according to claim 1, wherein the fraction of the extract of Geummokseo is a fraction fractionated with ethyl acetate.
The method of claim 1, wherein the 3-alpha,24-dihydroxy-urs-12-den-28-oic acid (3α,24-dihydroxy-urs-12-en-28-oic acid), pomolic acid ), maslinic acid, and corsolic acid compound is a cosmetic composition for skin whitening, characterized in that the compound is isolated from Geummokseo.

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