KR101866022B1 - Cosmetic composition for skin whitening containing Campylotropis hirtella extract with inhibitory activity of tyrosinase and melanin formation - Google Patents

Abstract

The invention kaempilro trophy's Nevsehir telra (Campylotropis hirtella) extract, or a cosmetic composition for skin whitening comprising the compound or a pharmaceutically acceptable salt thereof separated therefrom as an active ingredient having a tyrosinase and melanin synthesis inhibitory activity when the tie Specifically, the methanol extract of Campylotrophis hirtila root inhibited tyrosinase activity in a concentration-dependent manner. It was confirmed that the IC ₅₀ value was 20 μg / ml, and the compound isolated from the fraction of the extract Neorauflavane has an IC ₅₀ value of 30 nM for inhibition of monophenolase activity, which is 100 times more active than albutin and kojic acid, which are currently used as reference materials , And the IC ₅₀ value for the inhibition of the activity of dephenolase was 500 nM, and another compound The IC ₅₀ values for 3'-geranyl-5,7,2 ', 4'-tetrahydroxyisoflavanone were confirmed to be 2.9 μM and 128 μM for monophenolase and diphenolase, respectively. Also, the concentration of Campylo tropis hirtella extract In particular, Neorauflavane and 3'-geranyl-5,7,2 ', 4'-tetrahydroxyisoflavanone inhibited melanin synthesis, confirming that the I _C50 for inhibition of melanin synthesis was 13 μM and 42.3 μM, respectively, Neerauflavane and 3'-geranyl-5,7,2 ', 4'-tetrahydroxyisoflavanone isolated from Tella extract remarkably inhibit tyrosinase, monophenolase and diphenolase, and melanin synthesis, The composition containing the active ingredient is a cosmetic composition for skin whitening, and can be usefully used as a functional material for skin whitening and lightening.

Description

TECHNICAL FIELD The present invention relates to a cosmetic composition for skin whitening comprising Campylo tropis hirtela extract having an activity of inhibiting tyrosinase and melanin formation,

The present invention relates to a process for the production of < RTI ID = 0.0 > Campylotropis hirtella) extract or separated therefrom Neorauflavane and 3'-geranyl-5,7,2 ', 4' -tetrahydroxyisoflavanone, or a pharmaceutically acceptable cosmetic composition for skin whitening comprising the salt as an active ingredient and a health food .

Tyrosinase (EC 1.14.18.1) is the most important enzyme of melanin biosynthesis in plants, microorganisms and mammalian cells, and converts tyrosine to the key precursor of melanin biosynthesis, DOPA Quinone. The enzyme has a monophenolase function to convert L-tyrosine to DOPA (3,4-dihydroxyphenylalanine) and a diphenolase (DOPA) to convert L-dopa to L-dopaquinone diphenolase function (J. Appl. Microbiol., 2006, 100, 219-232; Int. J. Biochem. Cell Biol., 2004, 36, 235-246). The dopaquinone created here is automatically converted to melanin without the aid of enzymatic action. Therefore, the tyrosinase inhibitor inhibits melanin biosynthesis.

Melanin is a black pigment found in animals, plants and microorganisms. It is not essential for growth or development, but it is a substance that enhances the survival and competitiveness of the environment. Melanin is a stable pigment among the pigments found in organisms and does not dissolve in almost all solvents and the process of melanogenesis occurs in melanosomes, the organelles of specially differentiated cells, melanocytes . In other words, the skin color is determined by the content and distribution of melanin, and is related to the number and distribution of melanosomes released into the outside of the cell after being produced in melanocytes. Melanin protects skin from ultraviolet light (J. Drugs Dermatol., 2004, 3, 668-678). However, it is known that hyperpigmentation and melanomas such as spots and freckles are caused by excessive production.

In addition, tyrosinase has been shown to be an important cause of neurodegenerative diseases such as Parkinson's disease by converting dopamine, which plays an important role in brain function, into melanin and by decreasing the concentration of dopamine (Neurotox. Res., 2003 , 5, 165-176).

Conventionally, p-methoxy phenol, hydroquinone, kojic acid or arbutin have been used as melanin production inhibitors. However, these inhibitors have a problem of exhibiting side effects such as weak activity or denaturing pigment cells to impair the inherent functions of the cells.

In addition, for the purpose of inhibiting melanin production, vitamin C and its derivatives have been used, but these also have a problem of low tyrosinase inhibitory activity.

Therefore, it is urgently required to develop a new inhibitor which can inhibit tyrosinase activity and melanin formation even with a small amount of cytotoxicity (J. Agric. Food Chem., 1991, 39, 1897-1901).

Campylotropis hirtella ) belongs to leguminosae, a subtropical plant grown in Yunnan, southern China, and has been used as a traditional medicinal plant in China. Since ancient times, this plant has been used in women's diseases including prostate diseases, prostate diseases and stomach cancer, and major active metabolites such as coumarin, flavanoid, lignan, tannin and triterpene have been identified (J. Agric. Food Chem., 2009 , 57, 6712-6719; Fitoterapia, 2014, 95, 220-228), in particular geranyl group conjugated flavanoids have been shown to inhibit immunological activity (Bioorg. Med. Chem. Lett., 2009 , 19, 3389-3391). In addition, WEN, Ping et al. Have disclosed the bioactivity of seven flavonoids isolated from Campylotrophis hirtela (Journal of Shenyang Pharmaceutical University 6, 2008, 007), and the tyrosinase and The inhibitory activity of melanin synthesis is not known to date.

Accordingly, the present inventors have made efforts to discover natural materials that can inhibit tyrosinase activity or melanin production, which are superior to the conventional melanin production inhibitors described above, while methanol extracts of Campylotrophis hirtena roots are in a concentration- And the IC ₅₀ (the half maximal inhibitory concentration) value was found to be 20 μg / ml. From the fraction of the extract, Neorauflavane and 3'-geranyl-5,7,2 , And 4'-tetrahydroxyisoflavanone. Neorauflavane has an IC ₅₀ value of 30 nM for the inhibition of monophenolase activity, which is higher than that of albutin and kojic acid, which are currently used as standard substances. Neorauflavane had an IC ₅₀ value of 500 nM for the inhibition of the dephenolase activity, and 3'-geranyl-5,7,2 ', 4'-tetrahydrox yisoflavanone showed IC ₅₀ values for monophenolase and diphenolase of 2.9 μM and 128 μM, respectively. In addition, Campylo tropis hirtena extract inhibited melanin synthesis in a concentration-dependent manner, especially Neorauflavane and 3'-geranyl -5,7,2 ', 4'-tetrahydroxyisoflavanone showed that the IC ₅₀ for inhibition of melanin synthesis was 13 μM and 42.3 μM, respectively, indicating that camphorotrophic Hirtena extract and Neorauflavane and 3'-geranyl -5,7,2 ', 4'-tetrahydroxyisoflavanone exhibits remarkable inhibitory activity against tyrosinase, monophenolase and diphenolase, and melanin synthesis, so that a composition containing it as an active ingredient is a skin whitening cosmetic The present invention has been completed.

It is an object of the present invention to provide a tyrosinase inhibitor and a method for inhibiting melanin production by using Campylotropis providing hirtella) extract or separated therefrom Neorauflavane and 3'-geranyl-5,7,2 ', 4' -tetrahydroxyisoflavanone, or a cosmetic composition for skin whitening comprising the pharmaceutically acceptable salts thereof as an active ingredient will be.

In order to achieve the above object, the present invention provides a cosmetic composition for skin whitening comprising the kaempilro's trophy Nevsehir telra (Campylotropis hirtella) extract as an active ingredient.

In addition, the present invention relates to a method for inhibiting the growth of Campylotropis The present invention provides a cosmetic composition for skin whitening comprising, as an active ingredient, a compound represented by the following general formula (1) or (2) or a pharmaceutically acceptable salt thereof, which is isolated from a fraction of hirtella extract.

[Chemical Formula 1]

,

,

(2)

.

.

In addition,

Campylotropis The present invention provides a health functional food for skin whitening comprising, as an active ingredient, a compound represented by the following general formula (1) or (2) or a pharmaceutically acceptable salt thereof, which is isolated from a hirtella extract or a fraction thereof.

[Chemical Formula 1]

,

,

(2)

.

.

In the present invention, < RTI ID = 0.0 > Campylotropis hirtella ) inhibited the tyrosinase activity in a concentration-dependent manner and the IC ₅₀ (the half maximal inhibitory concentration) value was 20 μg / ml. The extracts of Neorauflavane and Neorauflavane Separating 3'-geranyl-5,7,2 ', 4'-tetrahydroxyisoflavanone, Neorauflavane has an IC ₅₀ value of 30 nM for the inhibition of monophenolase activity, which is the current standard of arbutin albutin and kojic acid, and Neorauflavane had an IC ₅₀ value of 500 nM for the inhibition of dephenolase activity, and 3'-geranyl-5,7, The IC ₅₀ values for 2 ', 4'-tetrahydroxyisoflavanone were 2.9 μM and 128 μM, respectively, for monophenolase and diphenolase, respectively. In addition, Campylo tropis hirtena extract inhibited melanin synthesis in a concentration-dependent manner . In particular, Neorauflavane and 3'-geranyl-5,7,2 ', 4'-tetrahydroxyisoflavanone showed that the I _C50 for inhibition of melanin synthesis was 13 μM and 42.3 μM, respectively. Thus, Campylo tropis hirtela extract, Neorauflavane and 3'-geranyl-5,7,2 ', 4'-tetrahydroxyisoflavanone exhibit remarkable inhibitory activity against tyrosinase, monophenolase and diphenolase, and melanin synthesis, Can be usefully used as a skin whitening cosmetic composition.

1 is a diagram showing ¹ H-NMR and ¹³ C-NMR spectra of Neorauflavane.
2 is a diagram showing ¹ H-NMR and ¹³ C-NMR spectra of 3'-geranyl-5,7,2 ', 4' -tetrahydroxyisoflavanone.
FIG. 3 is a graph showing the activity of inhibiting tyrosinase by concentration of Campylo tropis hirtena extract.
FIG. 4 is a graph showing the tyrosinase (monophenolase) inhibitory activity of Neorauflavane and 3'-geranyl-5,7,2 ', 4'-tetrahydroxyisoflavanone.
Figure 5 shows the inhibitory activity of tyrosinase (diphenolase) of Neorauflavane and 3'-geranyl-5,7,2 ', 4'-tetrahydroxyisoflavanone.
Fig. 6 is a diagram showing the inhibition of tyrosinase (monophenolase) of Neorauflavane by Lineweaver-Burk plot. Fig.
7 is a diagram showing the inhibition of tyrosinase (diphenolase) of Neorauflavane by Lineweaver-Burk plot.
8 is a graph showing the inhibition of tyrosinase (monophenolase) of 3'-geranyl-5,7,2 ', 4'-tetrahydroxyisoflavanone by Lineweaver-Burk plot.
FIG. 9 is a graph showing the inhibition of tyrosinase (diphenolase) of 3'-geranyl-5,7,2 ', 4'-tetrahydroxyisoflavanone by Lineweaver-Burk plot.
FIG. 10 is a diagram showing the inhibition of tyrosinase of Neorauflavane according to pre-incubation time. FIG.
Fig. 11 is a graph showing the results obtained by measuring the concentration of Campylotropis hirtella ) extract of Neorauflavane, which is a compound of the present invention.
12 is a graph showing changes in the activity of inhibiting melanin synthesis by the concentration of 3'-geranyl-5,7,2 ', 4'-tetrahydroxyisoflavanone, which is a compound isolated from Campylo tropis hirtena extract.

Hereinafter, the present invention will be described in detail.

The present invention relates to a process for the production of < RTI ID = 0.0 > Campylotropis hirtella extract as an active ingredient.

The Campylo Trophis hirtena extract or fraction thereof is preferably, but not limited to, prepared by a process comprising the steps of:

1) extracting an extract solvent by adding to Campylotrophis hirtila;

2) filtering the extract of step 1);

3) concentrating the filtered extract of step 2) under reduced pressure and drying to prepare a Campylotrophic hirutella extract; And

4) the step of extracting with Campylo tropis hirtena extract of step 3) with an organic solvent to prepare the camphyl tropis hirtena fraction.

In the above method, it is preferable to use water, an alcohol or a mixture thereof in the extraction solvent of the step 1). As the alcohol, C ₁ to C ₂ lower alcohol is preferably used, and as the lower alcohol, ethanol or methanol is preferably used. As the extraction method, it is preferable to use shaking extraction, Soxhlet extraction or reflux extraction, but it is not limited thereto. The extraction solvent is preferably added in an amount of 1 to 10 times the amount of the dried camphor tropis hirtela extract, more preferably 4 to 6 times of the extraction solvent. The extraction temperature is preferably 20 占 폚 to 100 占 폚, more preferably 20 占 폚 to 40 占 폚, and most preferably room temperature, but is not limited thereto. The extraction time is preferably 10 to 48 hours, more preferably 15 to 30 hours, most preferably 24 hours, but is not limited thereto. The number of times of extraction is preferably 1 to 5 times, more preferably 3 to 4 times, and most preferably, 3 times, but is not limited thereto.

In the above method, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration in step 3), but it is not limited thereto. The drying is preferably performed under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, but not always limited thereto.

In addition, the present invention relates to a method for inhibiting the growth of Campylotropis The present invention provides a cosmetic composition for skin whitening comprising, as an active ingredient, a compound represented by the following general formula (1) or (2) or a pharmaceutically acceptable salt thereof, which is isolated from a fraction of hirtella extract.

[Chemical Formula 1]

,

,

(2)

.

.

In addition, the extract is characterized by inhibiting tyrosinase activity, wherein the compound inhibits tyrosinase activity, wherein inhibition of tyrosinase activity is inhibited by monophenolase monophenolase) or a diphenolase activity inhibitor. Further, the compound is characterized by inhibiting melanin synthesis.

The present invention includes all of the compounds represented by formula (1) or (2) as well as their pharmaceutically acceptable salts, prodrugs thereof, possible solvates, hydrates, racemates or stereoisomers thereof .

The present invention can be used in the form of a compound represented by the general formula (1) or (2) or a pharmaceutically acceptable salt thereof, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Derived from non-toxic organic acids, such as, for example, diesters, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinic acid, succinic acid, succinic acid, succinic acid, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluene sulfonate, chlorobenzene sulfoxide Sulfonate, methanesulfonate, propanesulfonate, naphthalene-1-sulphonate, naphthalene-1-sulphonate, , Naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention can be produced by a conventional method, for example, by dissolving the compound represented by the formula (1) or (2) in an excess amount of an acid aqueous solution, and then mixing the salt with a water- Followed by precipitation using nitrile. It is also possible to prepare the same by heating the compound represented by the general formula (1) or (2) and an acid aqueous solution or alcohol, and then evaporating the mixture or drying the precipitated salt by suction filtration.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. The corresponding silver salt is also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable salt (such as silver nitrate).

The pharmaceutical composition of the present invention may further contain commonly used excipients, disintegrants, sweeteners, lubricants, flavors and the like, and may be formulated into tablets, capsules, powders, granules, suspensions, Syrups, and other liquid preparations.

In a specific example of the present invention, it was confirmed that the concentration of 100 μg / ml of Campylo Trophis hirterula inhibited the activity of tyrosinase by 80% or more, and the IC ₅₀ value was 30 μg / ml concentration (see FIG. 3) ). In addition, Neorauflavane isolated from the fraction of Campylo tropis hirtela showed an IC ₅₀ value of 30 nM for monophenolase and 2.5 μM for 3'-geranyl-5,7,2 ', 4'-tetrahydroxyisoflavanone 4, Fig. 6 and Fig. 8).

In addition, Neorauflavane showed an IC ₅₀ value of 13 μM for melanin biosynthesis inhibition and 42 μM for 3'-geranyl-5,7,2 ', 4'-tetrahydroxyisoflavanone (see FIGS. 11 and 12).

Therefore, Neorauflavane and 3'-geranyl-5,7,2 ', 4'-tetrahydroxyisoflavanone, which are compounds isolated from Campylo tropis hirtela extract and its fractions, have inhibitory activity on tyrosinase and melanin biosynthesis, It can be usefully used as a cosmetic composition for cosmetics.

The skin whitening cosmetic composition may be, but not limited to, lotion, ointment, gel, cream, patch, or spray. In preparing a composition for the improvement of skin beauty containing the camphylophyll hysterella extract of the present invention or a fraction thereof and a compound isolated therefrom, the composition for improving skin beauty, which is usually contained, An extract, or a fraction thereof, and a compound isolated therefrom may be added in an amount of 3 to 30 parts by weight, preferably 5 or 20 parts by weight.

In addition, the composition of the present invention may further contain, in addition to the camphyl tropis hydrera extract of the present invention or its fractions and the compounds isolated therefrom, a fatty substance, an organic solvent, a solubilizing agent, a thickening agent and a gelling agent, a softening agent, Stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, barrier agents, wetting agents, essential oils, dyes, pigments , Hydrophilic or lipophilic active agents, lipid vesicles or any other ingredient conventionally used in compositions for the improvement of skin aesthetics.

In addition, the components can be introduced in amounts commonly used in the dermatology field.

In addition,

Campylotropis The present invention provides a health functional food for skin whitening comprising, as an active ingredient, a compound represented by the following general formula (1) or (2) or a pharmaceutically acceptable salt thereof, which is isolated from a hirtella extract or a fraction thereof.

[Chemical Formula 1]

,

,

(2)

.

.

The extract inhibits tyrosinase activity, and the compound is characterized by inhibiting the activity of monophenolase or diphenolase.

In a specific embodiment of the present invention, the Campylo tropis hirtena extract inhibited the activity of tyrosinase by 80% or more, and the fractions of Neorauflavane and 3'-geranyl-5,7,2 ', 4'- tetrahydroxyisoflavanone showed monophenolase and diphenolase inhibitory activity and inhibited melanin biosynthesis.

Therefore, Neorauflavane and 3'-geranyl-5,7,2 ', 4'-tetrahydroxyisoflavanone, which are compounds isolated from Campylo tropis hirtena extract and its fractions, have inhibitory activity on tyrosinase and melanin biosynthesis, It can be usefully used as an effective ingredient of whitening health functional foods.

The health functional food of the present invention can be used as it is, or can be used in combination with other food or food ingredients, and can be suitably used according to a conventional method, as the above-mentioned Campylotrophis hirtila extract, or a fraction thereof, a compound isolated therefrom.

There is no particular limitation on the kind of the food. Examples of the food include functional mixed tea, dairy product, various kinds of soup, beverage, and vitamin complex, and include health functional foods in a conventional sense.

The camphyllofus hirtela extract according to the present invention, or a fraction thereof, and a compound isolated therefrom, can be added directly to food or used together with other food or food ingredients, and can be suitably used according to conventional methods. The amount of the active ingredient to be mixed may be suitably determined depending on the purpose of use. Generally, the amount of the camphyllofus hirtela extract, or a fraction thereof, and the compound isolated therefrom may be 0.01 to 15 wt% The health beverage composition may be added at a ratio of 0.02 to 5 g, preferably 0.3 to 1 g, based on 100 ml. However, in the case of long-term intake intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

The health functional beverage composition of the present invention has no particular limitation on the components other than the above-mentioned camphyla tropis hydrera extract, or a fraction thereof, and a compound isolated therefrom as an essential ingredient in the indicated ratios, A flavoring agent or a natural carbohydrate as an additional ingredient. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above .

In addition to the above-mentioned foods, the food of the present invention may contain flavors such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the inventive Campylotrophis hirtena extract, or its fractions, and the compounds isolated therefrom, may contain flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components may be used independently or in combination. Although the proportion of such additives is not so critical, it is common to choose from the range of 0 to about 20 parts by weight per 100 parts by weight of the camphor tropis hirtena extract of the present invention, or its fractions, the compounds isolated therefrom.

Hereinafter, the present invention will be described in detail with reference to Examples, Experimental Examples and Production Examples.

However, the following examples, experimental examples, and production examples are for illustrative purposes only, and the present invention is not limited by the following examples, experimental examples, and production examples.

< Example  1> Campylo Trophis Hirtela ( Campylotropis hirtella ) Preparation of extract

To prepare Campylo trophis hirutella extract, 0.5 kg of shredded Campylotrophis hirterella root (Yunnan Province, China) was crushed and extracted repeatedly 3 times for 1 week at room temperature with 5 L of methanol. The Campylo tropis hirtela methanol extract obtained through the above procedure was concentrated under reduced pressure to obtain 58 g of a dark brown crude extract (yield: 11.6%).

< Example  2> Campylo Trophis Hirtela Fraction  And separation and purification of compounds exhibiting activity

In order to separate and purify the compound exhibiting activity from the methanol extract obtained using the method of Example 1, the product was purified by silica gel chromatography (230: 1) using hexane and ethyl acetate mixed solvent (50: 1 -> 1: -400 mesh) was performed to obtain five fractions (AE). Among them, fraction C (3.2 g) was subjected to reverse phase chromatography using a mixed solvent of methanol and water, and 30 fractions (C1-C30) were obtained. Then, the fraction (C11-C15, 520 mg) was subjected to reverse phase chromatography using a mixed solvent of methanol and water in the same manner as described above to thereby isolate Neorauflavane (28 mg) pure. The fractions (C17-C21, 920 mg) were prepared as described above in 90% methanol solvent using Sephadex (LH-20), and 3'-geranyl-5,7,2 ', 4'- hydroxyisoflavanone (96 mg) was isolated pure.

("Active indicator compound" has been modified to "Compound" to comply with the patent law.)

&Lt; 2- 1> Campylo Trophis Hirtela From the fraction  Structural analysis of isolated compounds

The structure of the two kinds of compounds separated in Example 2 above can be analyzed by nuclear magnetic resonance spectroscopy such as ¹ H-NMR (500 MHz), ¹³ C-NMR (125 MHz), COZY, HMQC, and HMBC, , a polarizer and a circle saekbeop (Circular dichroism: CD) Neorauflavane and 3'-geranyl-5,7,2 'for the use was performed ^{by, 1 H-NMR (500 MHz} ), 13 C-NMR (125 MHz), The spectral results of 4'-tetra-hydroxyisoflavanone are shown in FIG. 1 and FIG. 2, and the analyzed results and the respective formulas are as follows:

Neorauflavane; colorless powder; ^{HREIMS [M] + 354.1467 (calcd} for C 21 H 22 O 5 354.1467); _{[α] D = +3.8 (c} 0.55, in CHCl 3); ^{1 H-NMR (500 MHz,} CDCl 3) δ 1.39 (3H, s, H-8 "), 1.40 (3H, s, H-7"), 2.78 (2H, dd, J = 15.95, 10.60 Hz, H -4), 2.96 (2H, dd , J = 16.00, 3.90 Hz, H-4), 3.40 (1H, br s, H-3), 3.72 (3H, s, OCH 3), 3.98 (2H, t, J = 20.10, 10.05 Hz, H -2), 4.26 (1H, d, J = 9.85 Hz, H-2), 5.51 (1H, d, J = 9.90, H-5 "), 6.18 (1H, s, H-8), 6.26 (1H , br s, H-3 '), 6.31 (1H, d, J = 7.70 Hz, H-5'), 6.51 (1H, d, J = 9.85 Hz, H-4 " ), 6.87 (1H, d, J = 8.25Hz, H-6 &apos;). ^{13 C-NMR (125 MHz,} CDCl 3) δ 155.32 (C-4 '), 155.28 (C-8a), 154.78 (C-2'), 154.11 (C-5), 152.64 (C-7), 128.26 (C-8), 128.15 (C-5), 119.87 (C-4a), 116.95 ), 103.22 (C-6 ' ), 100.76 (C-3'), 76.14 (C-6 "), 69.83 (C-2), 61.44 (OCH 3), 31.23 (C-3), 27.77 (C- 7 &quot;), 27.60 (C-8 &quot;), 25.33 (C-4).

3'-geranyl-5,7,2 ', 4'-tetrahydroxyisoflavanone; pale yellow oil; ^{HREIMS [M] + 424.1885 (calcd} for C 25 H 28 O 6 424.1886); ¹ H-NMR (500 MHz, CDCl ₃ ) ? 1.51 (3H, s, H-9 "), 1.58 (3H, s, H- 2H, m, H-5 " ), 2.11 (2H, m, H-6"), 3.39 (2H, d, J = 6.59 Hz, H-1 "), 3.92 (1H, t, J = 8.32, 4.16 Hz, H-3), 4.60 (2H, dd, J = 11.67, 4.39 Hz, H-2), 4.73 (2H, dd, J = 11.72, 4.47 Hz, H-2), 4.97 (1H, br s, H-7 "), 5.17 (1H, t, J = 12.71, 6.29 Hz, H-2"), 5.86 (1H, br s, (1H, d, J = 8.08 Hz, H-5 '), 7.08 (1H, d, J = 8.27 Hz, H-6'). ^{13 C-NMR (125 MHz,} CDCl 3) δ 197.56 (C-4), 165.86 (C-7), 165.51 (C-5), 163.59 (C-8a), 155.76 (C-4 '), 154.42 ( C-2 '), 139.61 (C-3 "), 132.41 (C-8"), 125.95 3 '), 115.43 (C-1'), 109.07 (C-5 '), 102.32 (C-4a), 97.32 (C-6), 95.80 C-3), 40.09 (C-5 "), 26.78 (C-1"), 26.03 ").

< Experimental Example  1> Campylo Trophis Hirtela  Of extracts and compounds Tairosineiz (tyrosinase) inhibitory activity measurement

In order to measure the tyrosinase inhibitory activity of the extracts and compounds carried out according to the methods described in <Example 1> and <Example 2>, the following experiment was conducted.

Specifically, the inhibitory effect of tyrosinase was measured by the method of Jeong et al. (J. Agric. Food Chem., 2009, 57, 1195-1203). L-tyrosine (L-tyrosine), L-DOPA (diphenolase) and the like were purchased from Sigma-Aldrich Co. (USA) And Spectra Max M3 Multi-Mode Microplate Reader (Molecular Device, USA). The samples used for the measurement were obtained by the methods described in Examples 1 and 2, and DMSO was added to the samples to obtain stock solutions of 10,000 μg / ml of the extract and 10 mM of the compounds. And they were diluted to suit the purpose of the experiment.

Specifically, add 165 μl of 0.25 M phosphate buffer (pH 6.8) and 20 μl of 1.8 mM L-tyrosine (monophenolase) or 3.6 mM L-DOPA (diphenolase) in buffer to a 96-well plate and measure 5 μl Samples were added and reacted at 37 ° C for 10 minutes. Add 10 μl of enzyme (tyrosinase, 260 unit / ml) dissolved in the buffer solution to the prepared well plate, and measure the dopachrome generated at the absorbance of 475 nm with a Microplate Reader instrument after further reaction for 15 minutes. Respectively. The inhibitory activity of the enzyme was determined by interpolation by the inhibition ratio or the concentration inhibiting 50% by IC ₅₀ value. The inhibition ratio was calculated as follows (Equation 1).

As a result, as shown in Fig. 3, the Campylotrophis hirtila extract inhibited tyrosinase activity in a concentration-dependent manner. In addition, their inhibitory activity evaluation showed IC ₅₀ at 30 μg / ml.

The isolated compound 3'-geranyl-5,7,2 ', 4'-tetrahydroxyisoflavanone exhibited IC ₅₀ values of 2.9 μM in the monophenolate substrate and 128 μM in the diphenolase substrate.

In addition, the separated compound Neorauflavane showed an IC ₅₀ value of 30 nM in a monophenolate substrate and 500 nM in a diphenolase substrate, and this value was found to be higher than kojic acid (Table 1, Fig. 4, and Fig. 5), which is 50 to 400 times or more higher than that of (monophenolase, IC ₅₀ = 13.2 μM), (diphenolase, IC ₅₀ = 26.5 μM).

compound L-tyrosine L-DOPA IC ₅₀ (mM) The inhibition type ( K _i , mM) IC ₅₀ (mM) The inhibition type ( K _i , mM) Neorauflavane 0.03 ± 0.006
Competitive (0.019)
0.5 ± 0.03 Competitive (0.13) 3'-geranyl-5,7,2 ', 4'-tetrahydroxyisoflavanone 2.9 ± 0.3 Competitive (1.8) 128.2 ± 0.5 Competitive (76.4)
Kojic acid 13.2 ± 0.8 Do not measure 26.5 ± 1.6 Do not measure

< Experimental Example  2> Campylo Trophis Hirtela From the fraction  Depending on discrete compound treatment Tairosineiz  Analysis of inhibition mechanism

Neorauflavane and 3'-geranyl-5,7,2 ', 4'-tetra-hydroxyisoflavanone of the present invention according to the results of Example 3 above inhibited the activity of tyrosinase in any form The Lineweaver-Burk plot was used to identify the foliage.

Specifically, the results of the above Example 1 were used to analyze spectra photoluminescence values of reactions occurring in reactants due to various concentrations of substrate and various concentrations of inhibitors. Data analysis was performed with a slope of change in emission value for 10 minutes, and the change in emission value of each section was measured every 30 seconds and evaluated by Lineweaver-Burk plot.

As a result, as shown in FIG. 6 to FIG. 9, when the compound isolated from Campylo tropis hirtela was treated, the concentration of the inhibitor in the Lineweaver-Burk plot for both of the two substrates (monophenolase and diphenolase) (1 / [v]) linear velocities at 1 / v (y-axis). Therefore, Vmax is not changed and Km is confirmed to be a typical competitive inhibitor.

< Experimental Example  3> Campylo Trophis From Hirtela  Depending on discrete compound treatment Taiyoshinaizu  Time-dependent inhibitory activity measurement

Time-dependent inhibitory activity and progression curves were measured using 250 units of tiresize, L-tyrosine (monophenolase), and L-DOPA (diphenolase) as substrates to 0.25 M potassium phosphate buffer 0.0 &gt; 37 C. &lt; / RTI &gt; Enzyme activity was measured at 405 nm using a visible light-ultraviolet spectrophotometer for 10 minutes. The time constant related rate constants were determined by measuring the concentration of the substrate and the concentration of the inhibitor for 10 minutes at intervals of 30 seconds. Data analysis was carried out using a nonlinear regression program (Sigma plot, Spcc Inc, Chicago, Ill., USA) and the slow binding inhibition model analysis, which is an important parameter for identifying inhibitory activity, Respectively.

As a result, as shown in Fig. 10A, the compound Neorauflavane showed a typical time-dependent inhibition pattern with an increase in activity according to the pre-incubation time, and the initial velocity Vi increased with increasing concentration of the inhibitor, And the steady-state rate (Vs) decreased (FIG. 10A). 10B, time-dependent inhibition patterns were also observed when the inhibitor concentration was varied and the extent of inhibition after pre-culture was measured. In addition, the initial rate (K _obs ) according to the Neorauflavane reaction time was observed to show a simple reversible slow binding inhibition pattern showing a linear pattern (R ² = 0.98) according to the compound concentration change (FIG. 10B).

< Experimental Example  4> Campylo Trophis From Hirtela  Analysis of the change of melanin content by treatment of isolated compounds

Neorauflavane and 3'-geranyl-5,7,2 ', 4'-tetrahydroxyisoflavanone, compounds isolated from the Campylotrophsiridae fraction, inhibit the pigmentation induced by α-MSH (melanocyte-stimulating hormone) B16F10 cells were treated with [alpha] -MSH and the above compounds and incubated for 72 hours.

As a result, when the α-MSH alone was treated as shown in FIGS. 11 and 12, the cell pellet changed to black, whereas Neorauflavane or 3'-geranyl-5,7,2 ', 4'-tetrahydroxyisoflavanone , The color of the pellets changed from black to white. That is, the compound inhibits the pigmentation induced by? -MSH. In particular, it was confirmed that Neorauflavane showed about 200-fold higher inhibition rate than kojic acid, which is the control, and that the whitening activity is more remarkable than 3'-geranyl-5,7,2 ', 4'-tetrahydroxyisoflavanone (Table 2 ).

compound Melanin synthesis
IC ₅₀ (mM) Cytotoxicity
LD ₅₀ (mM) Neorauflavane 13.0 ± 1.2 104.0 ± 1.8 3'-geranyl-5,7,2 ', 4'-tetrahydroxyisoflavanone 42.3 ± 1.6 240.6 ± 2.9 Kojic acid ^* > 500 > 500

< Experimental Example  5> Campylo Trophis Hirtela From the fraction  Cell viability analysis by treatment of isolated compounds

To confirm the cytotoxicity of Neorauflavane and 3'-geranyl-5,7,2 ', 4'-tetrahydroxyisoflavanone, compounds isolated from Campylo tropis hirtela fractions, B16F10 cells were treated with various concentrations of Neorauflavane or 3'-geranyl- The cell viability was determined by MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) analysis using 5,7,2 ', 4'-tetrahydroxyisoflavanone. Respectively.

As a result, as shown in Table 2, when Neorauflavane or 3'-geranyl-5,7,2 ', 4'-tetrahydroxyisoflavanone was treated, the cell lethal dose (LD ₅₀ ) value was 240.6 μM for Neorauflavane, geranyl-5,7,2 ', 4'-tetrahydroxyisoflavanone 104 μM (Table 2).

< Manufacturing example  1> Manufacture of health functional foods

<1-1> Solar  Produce

Brown rice, barley, glutinous rice, and yulmu were alfalted by a known method, dried and dispersed, and granulated to a powder having a particle size of 60 mesh.

Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a conventional method, and then they were prepared into powder having a particle size of 60 mesh by a pulverizer.

The camphylate trophethira extract of the present invention or its fraction was concentrated under reduced pressure in a vacuum concentrator, dried by spraying, and dried with a hot air drier, and the resulting dried product was pulverized to a size of 60 mesh with a pulverizer to obtain a dried powder.

The above-prepared cereals, seeds and camphor tropis hydrera extract or fractions thereof were prepared in the following proportions:

(30 parts by weight of brown rice, 15 parts by weight of yulmu, 20 parts by weight of barley)

Seeds (7 parts by weight of perilla, 8 parts by weight of black beans, 7 parts by weight of black sesame seeds)

(3 parts by weight) of the camphyl tropis hydratella extract of the present invention or its fraction,

(0.5 part by weight), and rhizome (0.5 part by weight).

<1-2> Manufacture of health drinks

The camphyl tropis hydratella extract of the present invention

Or its fractions of 100 mg

Citric acid 100 mg

100 mg of oligosaccharide

2 mg of plum concentrate

100 mg taurine

Purified water was added to 500 ml

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 DEG C for about 1 hour. The resulting solution was filtered to obtain a sterilized 1 liter container which was sealed and sterilized, &Lt; / RTI &gt;

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

< Manufacturing example  2> Preparation of composition for improving skin beauty

<2-1> Preparation of cream

The Campylo tropis hirtena extract of the present invention or

Its fraction 4.6 parts by weight

Cetostearyl alcohol 2.8 parts by weight

Beeswax 2.6 parts by weight

Stearic acid 1.4 parts by weight

Glycerin monostearate 2 parts by weight

&Lt; tb &gt; &lt; tb &gt;

Sorbitol sesquioleate 1.4 parts by weight

Jojoba oil 4 parts by weight

Squalane 3.8 parts by weight

Polysorbate 60 1.1 parts by weight

Macadia oil 2 parts by weight

Tocopheryl acetate 0.2 part by weight

Methylpolysiloxane 0.4 part by weight

Ethylparaben 0.1 part by weight

Propylparaben 0.1 part by weight

Euxyl K-400 0.1 part by weight

1,3-butylene glycol 7 parts by weight

Methylparaben 0.05 part by weight

Glycerin 6 parts by weight

d-Pandenol 0.2 part by weight

Triethanolamine 0.2 part by weight

pt 41891 0.2 part by weight

pH ₂ O 46.05 parts by weight

<2-2> Production of Lotion

The Campylo tropis hirtena extract of the present invention or

3.5 parts by weight of the fraction

Cetostearyl alcohol 1.6 parts by weight

Stearic acid 1.4 parts by weight

Glycerin monostearate of pro-type 1.8 parts by weight

&Lt; tb &gt; &lt; tb &gt;

Sorbitol sesquioleate 0.6 parts by weight

Squalene 4.8 parts by weight

Macadia oil 2 parts by weight

Jojoba oil 2 parts by weight

Tocopheryl acetate 0.4 part by weight

0.2 part by weight of methylpolysiloxane

Ethylparaben 0.1 part by weight

Propylparaben 0.1 part by weight

4 parts by weight of 1,3-butylene glycol

0.1 part by weight of methylparaben

Xanthan gum 0.1 part by weight

Glycerin 4 parts by weight

d-Pandenol 0.15 part by weight

Allantoin 0.1 part by weight

Cargar (2% aq. Sol) 4 parts by weight

Triethanolamine 0.15 part by weight

Ethanol 3 parts by weight

pt 41891 0.1 part by weight

pH ₂₀ 48.3 parts by weight

Claims (10)

delete delete delete A cosmetic composition for skin whitening comprising, as an active ingredient, a compound represented by the following formula (1) or (2) or a cosmetically acceptable salt thereof isolated from a fraction of Campylotropis hirtella extract:
[Chemical Formula 1]

,
(2)

.
The cosmetic composition for skin whitening according to claim 4, wherein the compound inhibits tyrosinase activity.
[Claim 6] The cosmetic composition for skin whitening according to claim 5, wherein the activity inhibition of tyrosinase is inhibited by monophenolase or diphenolase activity.
The cosmetic composition for skin whitening according to claim 4, wherein the compound inhibits melanin synthesis.
A skin-whitening health functional food containing, as an active ingredient, a compound represented by the following formula (1) or (2) or a pharmaceutically acceptable salt thereof isolated from a fraction of Campylotropis hirtella extract:
[Chemical Formula 1]

,
(2)

.
9. The skin care whitening health food according to claim 8, wherein the extract inhibits tyrosinase activity.
The health functional food for skin whitening according to claim 8, wherein the compound inhibits the activity of monophenolase or diphenolase.

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