KR101042005B1 - Compound from Lindera erythrocarpa, method for isolating thereof, and composition for skin whitening comprising the same - Google Patents

Abstract

The present invention relates to a compound derived from a birch tree, a method for separating the same, and a composition for skin whitening containing the same, and more particularly, a compound separated from a solvent fraction of an extract of Lindera erythrocarpa , a method for separating the same, and a compound containing the compound. The present invention relates to a composition for skin whitening that can suppress melanin biosynthesis.

Phenylpropane compounds, birch, extracts, solvent fractions, whitening, melanin, tyrosinase

Description

Compound from deciduous tree, isolation method thereof, and composition for skin whitening containing same {Compound from Lindera erythrocarpa, method for isolating etc, and composition for skin whitening comprising the same}

The present invention relates to a compound derived from a birch tree, a method for separating the same, and a composition for skin whitening containing the same, and more particularly, a compound separated from a solvent fraction of an extract of Lindera erythrocarpa , a method for separating the same, and a compound containing the compound. The present invention relates to a composition for skin whitening that can suppress melanin biosynthesis.

Research on physiologically active substances from natural products is being actively conducted, and plant resources are widely used as treatment and prevention or health supplements for diseases. Natural antioxidants include α-tocopherol, vitamin C, carotenoids, flavonoids, and the like. These antioxidants are widely distributed in animals and plants, and many researches are plant-derived. Secondary metabolites derived from plants have been found to inhibit or eliminate the production of free radicals and free radicals to prevent cell damage from oxidation.

Most of the natural antioxidants reported to date are plant-derived and are known to be primarily polyphenolic compounds. Flavonoids, in particular, act to prevent oxidation of lipids, scavenging free radicals and oxidative stress. It is effective in preventing or delaying diseases, and is being used in many fields such as food, medicine, and cosmetics. In addition, melanin (melanin) is a high molecular natural pigment of phenols widely present in animals, plants, and microorganisms as an important defense means to protect the living body from ultraviolet rays.

Melanin improves viability against UV rays, dryness, and extreme temperatures, and improves the quality of coffee, tea, and tobacco.However, excessive melanin synthesis causes blemishes, freckles, and skin spots on the human body, promotes skin aging, and causes skin cancer. It is known to be involved. The biosynthesis of melanin pigment is controlled by several enzymes, including tyrosinase enzymes, among which tyrosinase is a dihydroxyindole after the initial biosynthesis process in which tyrosine is transferred to L-dopaquinone. Acts on the oxidation of Therefore, the search for inhibitors of tyrosinase activity is an important part of the development of the whitening agent, and as the tyrosinase inhibitors which are still known, hydroquinone, 4-hydroxyanisole, ascorbic acid derivatives, kojic acid, Azelaic acid, corticosteroids, retinoids, arbutin, catechins, 3,4,5-trimethoxy cinnamate thymol ester However, there are many problems in their safety and economical efficiency, there is a difficulty in using.

On the other hand, nitric oxide (NO) is a small, relatively unstable and toxic inorganic low-molecular radical, which is a non-induced NOS (constitutive NOS (c-NOS) and inducible NOS for nitric oxide synthase (NOS)). (inducible NOS, iNOS) have been reported. NO production by cNOS plays an important role in the regulation of homeostasis in vivo, but unlike cNOS, iNOS differs from lipopolysaccharide (LPS), interferon-γ (interferon-γ, IFN-γ), and interleukin-1β (interleukin-1β, IL-1β) and tumor necrosis factor-α (TNF-α) are stimulated by macrophages, vascular smooth muscle cells, endothelial cells, hepatocytes and cardiomyocytes. INOS expressed in these tissues generates a large amount of NO for a long time, causing inflammation and tumors, and tissue damage, genetic mutations, and nerve damage. Therefore, in order to prevent and treat various diseases such as inflammatory diseases, researches using natural materials rather than artificial substances in functional fields, functional cosmetics, and therapeutic agents have been actively conducted.

The tree, Lindera erythrocarpa , is a plant of the Lauraceae family , which is distributed in 1,500 species of 45 genera and 6 genera and 12 species in Korea. A birch tree is a mausoleum with light yellow flowers in April to May and a red fruit of about 8mm in September. It is a deciduous tree with a height of 5m that grows in warm parts of Japan, China, southern part of Korea. Dried berries have an unusual aroma and bitter taste, and are used in Japan as an analgesic for gastrointestinal drugs and neuralgia. In addition, it has been reported that the extracts of the leaves and fruits of the birch tree have antifungal activity, and the cyclopentadione compounds isolated from the birch tree have the anticancer effect. Five reported cyclopentenedione compounds are lucidone, methyllucidone, linderone, methyllinderone, and kanakugiol. . And related efficacy studies have reported anti-inflammatory, antibacterial, anti-cancer.

In view of the above, the present inventors have isolated and identified polymethoxy phenolic compounds from ethanol extracts thereof using Lindera erythrocarpa , which has antifungal activity and anticancer activity, to B16F10 mouse melanoma cells. The present invention was completed by confirming that the compound can be utilized as a useful resource for whitening and functional cosmetics by investigating melanin biosynthesis inhibitory activity.

It is an object of the present invention to provide a novel compound derived from a birch tree.

Another object of the present invention is to provide a method for separating the compound from the birch tree.

In addition, another object of the present invention is to provide a composition for skin whitening containing the compound as an active ingredient.

In one embodiment, the present invention provides a compound represented by the following formula (1).

In the present invention, the compound of Formula 1 may be separated from the birch tree.

In the present invention, the compound of Formula 1 may be separated from the peeling of the birch tree.

As another aspect, the present invention provides a method for separating the compound represented by the following formula (1) from the birch tree.

[Formula 1]

In a preferred embodiment, the method for separating the compound of Formula 1 may include the following steps:

Extracting the birch tree with a solvent selected from water, C ₁ to C ₄ lower alcohols, or a mixed solvent thereof to obtain a birch tree extract;

Fractionating the non-tree extract with hexane, methylene chloride, ethyl acetate and butanol to obtain each solvent fraction;

Fractionating the ethanol solvent fractions by primary silica gel column chromatography using hexane and ethyl acetate as the solvent gradient as elution solvents; And

Separating the first silica gel column chromatography fraction using a mixed solvent of hexane and ethyl acetate as the elution solvent secondary silica gel column chromatography to separate the novel polymethoxy phenol compound of formula (1).

In the present invention, the tree is a peeled portion of the tree.

In the present invention, the tree extract may be obtained by extraction with a solvent selected from C ₁ to C ₄ lower alcohols such as water, ethanol, methanol, or a mixed solvent thereof, preferably ethanol. In the present invention, the extract may include an extract obtained by an extraction treatment, a dilution or concentrate of the extract, a dried product obtained by drying the extract, or any one of these modifiers or purified products.

As an embodiment of the present invention, the extract may be obtained by immersing the pulverized tree obtained by hot-air drying and grinding the lumber in 70% ethanol, leaching by stirring at room temperature for 3 days, filtered and concentrated under reduced pressure. .

In the present invention, the solvent fraction of the non-wood extract is suspended from distilled water with the non-wood extract from non-polar solvent n -hexane ( n -hexane) ( n -Hex), methylene chloride (methylene chloride) (CH ₂ Cl ₂ ), ethyl acetate (ethyl acetate) (EtOAc), and n - by fractionation using butanol (n -butanol) (n -BuOH) can be obtained.

As an embodiment of the present invention, the solvent fraction of the birch tree extract is suspended in distilled water by distillation of the birch tree ethanol extract, n -hexane ( n -Hex), methylene chloride (CH ₂ Cl ₂ ), ethyl acetate from a non-polar solvent in a separatory funnel (EtOAc) and n -butanol ( n- BuOH) were fractionated, filtered and concentrated under reduced pressure to obtain a fraction for each solvent.

As another aspect, the present invention provides a composition for skin whitening containing the compound represented by the following formula (1) as an active ingredient.

[Formula 1]

In a preferred embodiment of the present invention, the composition of the present invention is a cosmetic composition.

The components included in the cosmetic composition of the present invention include components conventionally used in cosmetic compositions in addition to the compound of formula 1 as an active ingredient, and include, for example, conventional auxiliaries such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavorings. And a carrier. In addition, the cosmetic composition may further include a skin absorption promoting substance to enhance the effect.

The cosmetic composition of the present invention may be prepared in any formulation commonly prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing , Oils, powder foundations, emulsion foundations, wax foundations, sprays, and the like, but are not limited thereto. Specifically, it may be prepared in the form of a flexible lotion, nutrition lotion, nutrition 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 oils, vegetable oils, waxes, paraffins, starches, trachants, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc or zinc oxide may be used as carrier components. Can be.

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

When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

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

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

In a preferred embodiment of the present invention, the composition of the present invention is a pharmaceutical composition.

When the composition of the present invention is made into a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like, including but not limited to It doesn't work. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

The pharmaceutical composition of the present invention can be administered by various routes such as oral or parenteral, for example orally or transdermally. Preferably, however, it is applied by topical application during parenteral administration, more preferably by topical application.

Suitable dosages of the pharmaceutical compositions of the present invention may vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be. The dosage of the pharmaceutical composition of the present invention may be administered once or several times a day in an amount of 5 to 30 mg / kg, preferably 10 mg / kg, in the case of an oral dosage form. It is recommended to continue 1 month or more by applying once to 5 times a day in an amount of 1.0 to 3.0 ml per day on an adult basis.

The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. In this case, the formulation may be used in any form suitable for pharmaceutical preparations, including powders, granules, tablets, capsules, suspensions, emulsions, syrups, oral formulations such as aerosols, external preparations such as ointments, creams, and the like. It may further include.

In a preferred embodiment of the present invention, the composition of the present invention is a food composition.

When the composition of the present invention is prepared as a food composition, it includes not only the compound of formula 1 as an active ingredient, but also components commonly added in the manufacture of food, and include, for example, proteins, carbohydrates, fats, nutrients, seasonings and Contains flavors. Examples of the above carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And sugars such as conventional sugars such as polysaccharides such as dextrin, cyclodextrin and the like and xylitol, sorbitol, erythritol. As flavoring agents, natural flavoring agents (tauumatin, stevia extract (for example levaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used.

The present invention can provide a compound derived from a birch tree and a method for separating the same, and also by including the compound as an active ingredient can provide a composition for skin whitening showing a higher melanin inhibitory activity than the arbutin known as a whitening agent It has a very good effect.

Hereinafter, the configuration and effects of the present invention will be described in more detail with reference to examples, but these examples are merely illustrative of the present invention, and the scope of the present invention is not limited only to these examples.

Example 1 Preparation of Extracts from Leaves and Peel of the Tree and Separation of Solvent Fractions

The land plant sample used in this example was used as a land plant sample ( Linera erythrocarpa ), which was sold by the Jeju Hi-Tech Industrial Development Institute Extract Bank, and the solvent used for the extraction of the sample was Merk Co.'s product.

The raw leaves of the birch tree were washed with running water, and then dried at 40 ° C. for hot air for 3 days, and then pulverized with a grinder. 200 g of the dry powder sample was immersed in 70% ethanol and extracted by stirring for 24 hours at room temperature for 3 days, and the leachate was filtered through a filter. After the leaching filtration process was repeated three more times, the filtrate was concentrated under reduced pressure. Take 42 g of 60 g of the obtained extract (70% ethanol extract), suspend in distilled water, and separate with n -hexane ( n -Hex), methylene chloride (CH ₂ Cl ₂ ), ethyl acetate (EtOAc), n- Fractionation was performed using butanol ( n- BuOH), followed by filtration and concentration under reduced pressure to obtain 8.56 g of each solvent-based fraction n -hexane, 1.47 g of methylene chloride, 4.14 g of ethyl acetate, 10.2 g of n -butanol, and 14.58 g of water.

On the other hand, non-wood peeling was also dried and pulverized in the same manner as the leaves, and the solvent fraction for each polarity was obtained using 68.2 g of 70% ethanol extract obtained by extracting under the same conditions using 560 g of dry powder samples, each fraction was obtained.

Example 2 Isolation and Structural Identification of Compounds from Solvent Fractions

As a filler used in the separation process of this example, a normal-phase silica gel 60 (0.063-0.200 mm, Merck Co.) was used. The instrument used in the analysis was HPLC (Alliance2695, Waters Co.), XTerra ^® C18 3.5μm 4.6 × 100mm was used, and the detector used in the analysis was detected by waters 2998 model PDA . HPLC grade was used as the solvent and reagents in the experiment, and NMR (Nuclear Magnetic Resonance) was used as the instrument used for structural analysis. JNM-LA500 of Burker (German Co.) was used. , Methanol- d ₄ and chloroform- d ₁ were used.

Ten fractions were obtained by fractionation of methylene chloride fraction layer (2.5 g) of the non-tree leaves obtained in Example 1 under normal silica gel (hexane / ethyl acetate) (v / v) and solvent gradient conditions. Of these, compound 1 (21.8 mg) was obtained by recrystallization in fraction-6.

On the other hand, 8 fractions were obtained by fractionating methylene chloride fraction layer (7.0 g) of the birch bark obtained in Example 1 under normal silica gel (hexane / ethyl acetate) (v / v) and solvent gradient conditions. Chromatography was carried out using fraction-2 of the above fractions under conditions of normal silica gel (hexane / ethyl acetate) (3/2), to which compound 3 (59.6 mg) was obtained in fraction-2-2, and fraction-2- Compound 2 was obtained in 3.

Compounds 1 to 3 obtained as described above were analyzed by HPLC, NMR, (1D, 2D NMR), and LR / HR FAB-MS for compound analysis and structure confirmation.

As a result, Compound 1 was identified as lucidone of the cyclopentadione series of the formula (2). 1D and 2D NMR data of compound 1 are shown in Table 1 below.

[Formula 2]

1D and 2D NMR Data of Compound 1 location
(Position) Lucidon δ _c (mult) ^a δ _H (int, mult, J in Hz) HMBC (H → C) One 135.0 2 129.2 7.61 (1H, doublet of doublets, 7.5.3.0 Hz) C-4, C-6, C-7 3 111.8 7.39 (1 H, m ) C-1 4 130.9 7.39 (1 H, m ) C-1 5 128.9 7.39 (1 H, m ) C-1 6 129.2 7.61 (1H, doublet of doublets, 7.5.3.0 Hz) C-4, C-6, C-7 7 143.2 7.74 (1 H, d , 16.0 Hz) C-6, C-8, C-9 8 117.8 7.64 (1 H, d , 16.0 Hz) C-1, C-7, C-9 9 168.4 One' 103.1 2' 198.9 3 ' 171.2 3.94 (3H, s ) 4' 108.1 C-5 ' 5 ' 185.0 6 ' 59.0 5.82 (1H, s ) C-9, C-3 ' ¹ H NMR (500 MHz), ¹³ C NMR (125 MHz), CD ₃ OD

Compound 2 was identified as methyllinderone of Formula 3 of the same series. 1D and 2D NMR data of Compound 2 are shown in Table 2 below.

(3)

1D and 2D NMR Data of Compound 2 location
(Position) Methylrinderon δ _c (mult) ^a δ _H (int, mult, J in Hz) HMBC (H → C) One 135.8 2 128.5 7.58 (1H, doublet of 7.7 , 1.5 Hz) 3 129.1 7.35 (1 H, m ) 4 130.2 7.35 (1 H, m ) C-1 5 129.1 7.35 (1 H, m ) C-8 6 128.5 7.58 (1H, doublet of 7.7 , 1.5 Hz) 7 121.4 7.49 (1 H, d , 16.0 Hz) C-1, C-9 8 141.4 7.92 (1 H, d , 16.0 Hz) C-1, C-6, C-7, C-9 9 165.6 10 64.5 4.08 (3H, s ) C-9 One' 109.6 2' 187.4 3 ' 149.1 4' 148.0 5 ' 184.9 6 ' 60.1 4.17 (3H, s ) C-4 ', C-5' 7 ' 60.1 4.17 (3H, s ) C-4 ', C-5' ¹ H NMR (500 MHz), ¹³ C NMR (125 MHz), CD ₃ OD

Compound 3 is a derivative compound modified in the cyclopentadione series of Formula 1, and has been identified as a novel compound (N2) that has not been reported to date. 1D and 2D NMR data of compound 3 are shown in Table 3 below.

The IUPAC name of the novel compound is 1- (2-hydroxy-3,4,5,6-tetramethoxyphenyl) -1-methoxy-3-phenylpropane [1- (2-hydroxy-3,4,5 , 6-tetramethoxyphenyl) -1-methoxy-3-phenylpropane] and its generic name is jeju-erythrane.

[Formula 1]

Properties of the compound

1) IUPAC Name: 1- (2-hydroxy-3,4,5,6-tetramethoxyphenyl) -1-methoxy-3-phenylpropane

2) Color: Yellow

3) Incense: weak perishable odor

4) Solubility: organic solvents such as methanol, acetone and chloroform

5) Distribution only on the tree peel

6) Common Name: jeju-erythrane

1D and 2D NMR Data of Compound 3 location
(Position) New compound (jeju-erythrane) δ _c (mult) ^a δ _H (int, mult, J in Hz) HMBC (H → C) One 78.9 (d) 4.71 (1H, doublet, 8.5, 4.5) C-2, C-3, C-1 ' 2 37.8 (t) 1.98 (1 H, m)
2.26 (1 H, m) 3 32.8 (t) 2.65 (1H, ddd, 15.0, 9.0, 7.5)
2.82 (1H, ddd, 15.0, 9.5, 4.0) 1-OCH ₃ 57.5 (q) 3.34 (3H, s) C-1 One' 115.3 (s) - 2' 148.4 (s) - 3 ' 148.0 (s) - 4' 140.3 (s) - 5 ' 138.6 (s) - 6 ' 146.5 (s) - 3'-OCH ₃ 61.1 (q) 3.72 (3H, s) C-2 ' 4'-OCH ₃ 61.5 (q) 3.87 (3H, s) C-3 ' 5'-OCH ₃ 61.4 (q) 3.76 (3H, s) C-4 ' 6'-OCH ₃ 61.4 (q) 3.78 (3H, s) C-5 ' 2'-OH - 8.8 (1 H, s) C-1 ', C-5', C-6 ' One" 142.8 (s) - 2 "& 6" 129.4 (d) 7.22 (2H, double, 7.5) 3 "& 5" 129.2 (d) 7.27 (2H, t, 7.5) C-3, C-1 " 4" 126.6 (d) 7.16 (1H, t, 7.5) ¹ H NMR (500 MHz), ¹³ C NMR (125 MHz), acetone- d ₆
^a Determined by DEPT experiments.

The ethanol extracts of the birch leaves and the peels and the fractions for each solvent and the yields of the compounds of Formula 1 are shown in Table 4 below.

matter Yield (%) leaf Peel 70% Ethanol Extract 30.0 12.1 n -hexane fraction 14.3 7.3 Methylene chloride fraction 1.47 10.2 Ethyl acetate fraction 4.14 18.0 n -butanol fraction 10.2 26.9 Water fraction 14.5 43.4 Compound of Formula 1 - 0.71

As can be seen from Table 4, the compound of Formula 1 was not found in the leaf, but was observed as high as 0.71% in the peeling.

The single compound isolated from the birch tree was completely dissolved by adding a DMSO solvent, filtered, and used in subsequent experiments.

Experimental Example 1 Cytotoxicity Measurement of the Compound of Formula 1 by MTT Assay

In order to determine the effect on the cell viability of the B16F10 cell line using a novel compound which is currently unknown.

B16F10 mouse melanoma cells used in this experiment were distributed from the American Cell Line Bank (ATCC). B16F10 cells were treated at 37 ° C., 5% CO using 10% fetal bovine serum (FBS, Gibco), 1% Antibiotic-Antimycotic (Gibco), DMEM medium containing L-glutamine and sodium bicarbonate (Gibco). Cultured in ₂ cell culture phase.

B16F10 cells were inoculated with 2 × 10 ⁴ cells per well in a 24 well plate and incubated in a 37 ° C., 5% CO ₂ cell incubator for 24 hours, and then the samples were inoculated into each well of 5 μg / ml, 10 μg / ml, Treatments were performed at various concentrations up to 20 μg / ml and 30 μg / ml for 3 days and incubated for 72 hours. 200 μl of the MTT solution prepared at a concentration of 2 mg / ml was added thereto, and cultured for 4 hours under the same culture conditions to remove the medium, and the cells were washed twice with PBS. 200 μl of DMSO was added to each well, and the absorbance was measured at 570 nm with an ELISA reader (μQuant, USA).

As a result, as shown in Figure 1 when the cell survival rate of the control group to 100%, the compound of Formula 1 was slightly reduced or increased in the control group to almost 97 ~ 103% at 5, 10, 20, 30 ㎍ / ㎖ concentration. As compared with the control group, the concentrations of 5 μg / ml, 10 μg / ml, 20 μg / ml and 30 μg / ml were slightly different but did not show a significant change. It could be confirmed that it can be used as a whitening agent.

Experimental Example 2 Measurement of Melanin Inhibitory Activity of the Compound of Formula 1 of the Present Invention

Inoculate 2 x 10 ⁴ B16F10 cells per well into a 24 well plate and incubate in a 5% CO ₂ cell incubator for 24 hours at 37 ° C. Was washed twice with PBS. 500 μl of 1N NaOH was added to each well and dissolved at 56 ° C. for 30 minutes, and then absorbance was measured at 405 nm with an ELISA reader (μQuant, USA).

As a result, as shown in FIG. 2, the treatment of arbutin, known as a whitening agent, at 50 ㎍ / ml showed melanogenesis inhibition activity of 33.6%. Higher inhibitory activity of 19%, 32%, 51%, and 59% was obtained when treated to 20 μg / ml and 30 μg / ml. Therefore, the melanin inhibitory activity was about 2 times higher than that of the control arbutin, and showed a good result as a whitening agent in natural plants.

Experimental Example 3: Determination of mRNA expression inhibitory activity involved in the production of melanin of the compound of formula 1 of the present invention

B16F10 mouse melanoma cells used in this experiment were distributed from the American Cell Line Bank (ATCC). B16F10 cells were treated at 37 ° C., 5% CO using 10% fetal bovine serum (FBS, Gibco), 1% Antibiotic-Antimycotic (Gibco), DMEM medium containing L-glutamine and sodium bicarbonate (Gibco). Cultured in ₂ cell culture phase.

After culturing the cells were washed 2-3 times with PBS, total RNA extraction was separated using TRIzol-reagent (Invitrogen, USA). The cells were homogenized by addition of TRIzol-reagent, and then centrifuged (12,000 rpm, 15 min) by addition of chloroform. The same amount of isopropanol was added to the supernatant, centrifuged (12,000 rpm, 10 min) to precipitate RNA, washed with 75% diethylpyrocarbonate (DEPC) -treated ethanol, and dried and dissolved in DEPC-treated distilled water. RNA was quantified by measuring absorbance at 260 nm, and RNA having a value in the range of A260 / A280 nm in the range of 1.6-1.9 was used for the experiment. cDNA synthesis ^{Improm-Ⅱ TM cDNA kit (Promega} , USA) to the had, total RNA in 1 ㎍ oligo (dT) primer, dNTP (0.5 μM), 1 unit RNase inhibitor used and ^{Improm-Ⅱ TM reverse transcriptase (2} U The reaction was stopped by heating 25 ° C. for 5 minutes, 37 ° C. for 60 minutes, and 70 ° C. for 10 minutes. Polymerase chain reaction (PCR) was carried out to amplify tyrosinase, TRP-1, TRP-2, and β-actin from synthesized cDNA, 1 μl cDNA, 4 μM 5 'and 3' primer, 10 × buffer (10 mM Tris- HCl, pH 8.3, 50 mM KCl, 0.1% Triton X-100), 250 μM dNTP, 25 mM MgCl ₂ , 1 unit Taq polymerase (Promega, USA), mix to final 25 μl with tertiary distilled water and then Perkin-Elmer PCR was performed using a thermal cycler. At this time, PCR conditions were 94 ℃ / 30 seconds, 50 ~ 55 ℃ / 45 seconds, 72 ℃ / 45 seconds 20 ~ 25 times, and the product produced by PCR was subjected to electrophoresis on 1.2% agarose gel and stained with ethidium bromide. I identified a specific band.

Melanin cells are known to be involved in the process of NO produced in keratinocytes by UV light to increase melanin production, increase tyrosinase and TRP-1, and induce mRNA expression of tyrosinase activity. RT-PCR was performed to determine whether the melanin inhibitory effect of the single compounds isolated from the birch at the concentration that does not show toxicity in B16F10 mouse melanoma cells is due to the inhibition of mRNA expression. Inhibition of expression of TRP-1 gene other than tyrosinase, which is known as a major enzyme for melanin production, was confirmed.

As a result, as shown in FIG. 3, the expression levels of tyrosinase and TRP-1 mRNA were decreased in a concentration-dependent manner. Usually, in the process of melanin synthesis, TRP-2 is a carboxylated derivative of 5,6-dihydroxyindole-2-carboxylic acid (DHICA), which is a carboxylated derivative of dopachrome. Although it is reported that the function of converting the TRP-2 mRNA gene, TRP-2 mRNA expression was constant regardless of the concentration.

Therefore, the novel compounds isolated from the birch tree were found to inhibit melanin synthesis by reducing the mRNA expression of tyrosinase and TRP-1 irrespective of TRP-2 (FIG. 3). As a result, it was confirmed that the new compound isolated from the birch tree has excellent melanin production and tyrosinase inhibitory effect on B16F10 melanoma cells, and thus it has a useful value as a whitening-related functional material.

1 is a graph showing the effect of the compound of formula 1 derived from the tree of the present invention on the cell survival rate of B16F10 cells.

Figure 2 is a graph showing the effect of the compound of formula 1 derived from the tree of the present invention on the inhibition of melanogenesis in B16F10 cells.

Figure 3 shows the aspect of the compound of formula 1 derived from the tree of the present invention inhibits the expression of tyrosinase and TRP-1 gene in B16F10 cells. In this case, C means a control, Arb means arbutin.

Claims (11)

Compound represented by the following formula (1): [Formula 1]

The compound of claim 1, wherein the compound of Formula 1 is isolated from a birch tree. The compound of claim 1, wherein the compound of Formula 1 is separated from the peeling of the birch tree. Extracting the birch tree with a solvent selected from water, C ₁ to C ₄ lower alcohols, or a mixed solvent thereof to obtain a birch tree extract; Fractionating the non-tree extract with hexane, methylene chloride, ethyl acetate and butanol to obtain each solvent fraction; Fractionating the ethanol solvent fractions by primary silica gel column chromatography using hexane and ethyl acetate as solvent elution solvents; And A method of separating the compound of claim 1 from the birch tree comprising the step of performing the second silica gel column chromatography using the mixed solvent of hexane and ethyl acetate as the elution solvent. 5. The method of claim 4, wherein the birch tree is a peeled portion of the birch tree. Skin whitening composition containing a compound represented by the following formula (1) as an active ingredient: [Formula 1]

The composition for skin whitening according to claim 6, wherein the compound of Chemical Formula 1 is isolated from a birch tree. The composition for skin whitening according to claim 6, wherein the compound of Formula 1 is separated from the peeling of the birch tree. The composition for skin whitening according to any one of claims 6 to 8, wherein the composition for skin whitening is a cosmetic composition. The composition for skin whitening according to any one of claims 6 to 8, wherein the composition for skin whitening is a pharmaceutical composition. The composition for skin whitening according to any one of claims 6 to 8, wherein the composition for skin whitening is a food composition.

Images