KR101069907B1 - A composition for skin whitening comprising extract, fraction or compound from Lindera erythrocarpa - Google Patents

Abstract

The present invention relates to a composition for skin whitening containing extracts, fractions or compounds derived from a birch tree, and more particularly, an ethanol extract obtained by extracting birch trees with ethanol, a solvent fraction obtained therefrom or a compound purified therefrom. It relates to a composition for skin whitening comprising as an active ingredient.

Skin whitening composition containing extracts, fractions or compounds of the present invention is higher than Arbutin, which is known as a whitening agent, by containing as an active ingredient a tree extract, a solvent fraction thereof, or a cyclopentadione compound purified from it. It has a very good effect of showing melanin inhibitory activity.

Tree, Lindera erythrocarpa, Extract, Solvent fraction, Whitening, Melanin, Cyclopentadione

Description

A composition for skin whitening comprising extract, fraction or compound from Lindera erythrocarpa}

The present invention relates to a composition for skin whitening containing extracts, fractions or compounds derived from a birch tree, and more particularly, an ethanol extract obtained by extracting birch trees with ethanol, a solvent fraction obtained therefrom or a compound purified therefrom. It relates to a composition for skin whitening comprising as an active ingredient.

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, and flavonoids. These antioxidants are widely distributed in plants and animals, and many of them have been 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 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, thus preventing anti-aging, cancer and heart. It is effective in preventing or delaying diseases, and is being used in many fields such as food, medicine, and cosmetics. Melanin is also a polymeric natural pigment of phenols, which is widely present in animals, plants, and microorganisms as an important defense means of protecting the living body from ultraviolet rays.

Melanin improves viability against UV rays, dryness and extreme temperatures, and improves the quality of coffee, tea, tobacco, etc., but excessive melanin synthesis creates spots, freckles, 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 regulated by various enzymes including tyrosinase enzyme, among which tyrosinase acts on the oxidation of dihydroxyindole after the initial biosynthesis process of tyrosine as a substrate to L-dopaquinone. Therefore, the search for inhibitors of tyrosinase activity is an important part of the development of the whitening agent, and hydroquinone, 4-hydroxyanisole, ascorbic acid derivatives, kojic acid, azelaic acid, corticosteroid, retinoids, arbutin, catechin are known as tyrosinase inhibitors. , 3,4,5-trimethoxy cinnamate thymol ester, etc., but there are many problems in their safety and economic efficiency, so there is difficulty in using.

Lindera erythrocarpa ) is a plant of the family Lauraceae . It is known that 45 genera and 1,500 species are distributed worldwide, and 6 genera and 12 species are native to 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, EtOH extract extracted from the leaves and fruits of the birch tree and lucidone, a cyclopentenedione family, have been reported to have high anti-inflammatory effects (Wang et al., 2008). Cyclopentadione compounds isolated from the birch tree have anti-cancer effects. (Oh et al., 2005; Patent 10-0658519).

In view of the above, the present inventors have obtained ethanol extracts, sequential solvent fractions, and cyclpentadione compounds using Lindera erythrocarpa in Jeju Island to search for antioxidant activity, and to B16F10 mouse melanoma cells and RAW 264.7 cells. By treating melanin biosynthesis inhibitors by treatment, the present invention was completed by confirming that they are useful as useful resources for skin whitening functional cosmetics, foods or pharmaceutical compositions.

Accordingly, it is an object of the present invention to provide a composition for skin whitening containing an extract of a birch tree, a solvent fraction thereof, or a cyclopentadione compound separated and purified therefrom as an active ingredient.

In one embodiment, the present invention provides a composition for skin whitening containing an extract of a birch tree, a solvent fraction thereof or a cyclpentadione compound separated and purified therefrom as an active ingredient.

In the present invention, the birch tree extract includes water, ethanol, lower alcohols such as methanol or a solvent selected from a mixed solvent thereof, preferably extracted with ethanol. In addition, in this invention, the said extract shall contain the extract obtained by an extraction process, the dilution or concentrate of an extract, the dried material obtained by drying an extract, or any of these modifiers or refined 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 suspending the non-wood extract with distilled water and then from the 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 from the non-polar solvent n- hexane ( n -Hex), methylene chloride (CH ₂ Cl ₂ ), ethyl acetate (EtOAc) and n -butanol ( n -BuOH) were fractionated, filtered and concentrated under reduced pressure to obtain a fraction for each solvent.

In the present invention, as a skin lightening active cycle penta-dione compound separated and purified from the Item of expenditure tree quercetin (quercetin), quercitrin (quercitrin), car amp Ferrol -3- O - Lam nopi Llano side (kaempferol-3- O -rhamnopyranoside ), quercetin -3- O - arabinose furanyl furnace side (quercetin-3 -O -arabinofuranoside), caffeic acid ethyl ester (caffeic acid ethyl ester), cinnamic acid methyl ester (cinamic acid methyl ester), Lucy money (lucidone) , Methyl linderone and kanakugiol.

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 extract of the birch tree or solvent fractions thereof as active ingredients, and include, for example, antioxidants, stabilizers, solubilizers, vitamins, pigments and flavorings. Conventional adjuvants and carriers may be included. 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.

In the case where 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 as a carrier component. Especially, in the case of a spray, a mixture of chlorofluorohydrocarbons, propane / Propane 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, as an active ingredient, as well as the extract of the lumber tree or fractions thereof, it includes components that are commonly added during food production, for example, proteins, carbohydrates, fats, nutrients, seasonings. And flavoring agents. 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 the flavoring agent, natural flavoring agents (tauumatin, stevia extract (for example rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used.

Skin whitening composition containing extracts, fractions or compounds of the present invention is higher than Arbutin, which is known as a whitening agent, by containing as an active ingredient a tree extract, a solvent fraction thereof, or a cyclopentadione compound purified from it. It has a very good effect of showing melanin inhibitory activity.

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: birch extract manufacture

Lindera , a plant sample used in this example ( Lindera erythrocarpa ) was used by Jeju Hi-Tech Industrial Development Institute Extract Bank.

First, Lindera erythrocarpa Makino. 200 g of the dried sample was immersed in 70% ethanol and stirred for 3 days at room temperature. The leachate was filtered through a filter, the leaching filtration was repeated three more times, and the filtrate was concentrated under reduced pressure. g of ethanol extract were obtained.

Example  2: derived from the tree extract Solvent fraction  detach

Solvents used in the extraction of the sample in this example was a product of Merk Co., Junsei Co., Hyman Co.

Solvent fractions for the ethanol extracts of the birch tree were performed as follows.

First, 42 g of 60 g of the 70% ethanol extract prepared in Example 1 was suspended in distilled water, and then suspended in distilled water, using n- hexane ( n- Hex) and methylene chloride (CH ₂ Cl). ₂ ), ethyl acetate (EtOAc), n -butanol ( n -BuOH), fractions were collected, filtered and concentrated under reduced pressure. N- hexane 8.56 g, CH ₂ Cl ₂ 1.47 g, EtOAc 4.14 g, n- BuOH 10.2 g, H ₂ O 14.58 g were obtained (see FIG. 1).

HPLC analysis was performed on each extract and fraction obtained as above. The results are shown in FIGS. 2 to 7.

In subsequent experiments, the powdered hexane, methylene chloride, and ethyl acetate fractions were dissolved in 100% ethanol, but the butanol and water fractions were mixed with 100% ethanol and 1 × Phosphate Buffered Saline (PBS, pH 7.4) in a 1: 1 ratio. It was added completely to dissolve and then filtered.

Example  3: of birch ethanol extract From solvent fractions  Isolation and Structure Identification of Compounds

The ethyl acetate fractionation layer (4.0 g) was used to separate the fractions using methylene chloride, ethyl ether, ethyl acetate, and acetone using celite. Among them, ethyl ether fractions were used, and silica gel, chloroform and methanol were used as a developing solvent. Of these, fraction-5 was used to obtain compound 1 from fraction-1 and fraction-2 from fraction-3 using pure silica gel, chloroform and methanol. Fraction-2 obtained Compound 3 and Compound 4 using a 30% aqueous methanol solution using Prep-HPLC.

Ten fractions were obtained using a methylene chloride fraction layer (2.53 g) using pure silica gel, hexane and ethyl acetate as solvent gradient conditions. Using fractional silica gel with fraction-2, hexane and ethyl acetate were purified using a developing solvent to obtain compound 5. Compound 5 was obtained using fractional silica gel using chloroform and methanol as developing solvent. Fraction 6 obtained compound 7 by recrystallization, and fraction-7 obtained compound 8 using recrystallization. Fraction-8 was prepared using chloroform and methanol as a developing solvent using pure silica gel, and two fractions were obtained. Among them, compound 9 was obtained from fraction-8-2. The nine compounds obtained were identified by HPLC analysis and NMR. It was confirmed that the separation.

Finally, 200 g of the leaves of the tree were extracted with 70% ethanol to obtain 60 g of the extract, from which nine compounds were purified purely.

Yields for the separated fractions and compounds are shown in Table 1 below.

Yield of the present invention birch tree extract and each fraction Ext. solvent Yield (%) n- hexane Fr. 20.400 CH ₂ Cl ₂ Fr. 6.000 EtOAc Fr. 9.800 n -butanol Fr. 24.200 H ₂ O Fr. 34.700 Compound 1 0.600 Compound 2 0.060 Compound 3 0.002 Compound 4 0.720 Compound 5 0.020 Compound 6 0.030 Compound 7 0.240 Compound 8 0.090 Compound 9 0.110

¹ H, ¹³ C, COSY, HMQC, and HMBC spectra were obtained using a nuclear magnetic resonance apparatus (Bruker Co. 500MHz, NMR), and the structures were analyzed by comprehensive analysis of these spectra to determine high performance liquid chromatography (HPLC). Quantitative analysis was performed.

High performance liquid chromatography results and ¹ H and ¹³ C-NMR spectra of each compound are shown in FIGS. 8 to 25.

As a result, Compound 1 was identified as quercetin of Formula 1.

Compound 2 was identified as quercitrin of the formula (2).

Compound 3 to a car amp Ferrol -3- O of Formula 3 was identified as indigo nopi Llano side (kaempferol-3- O -rhamnopyranoside).

Compound 4 to quercetin of formula 4 -3- O - was identified as arabinose furanyl furnace side (quercetin-3 -O -arabinofuranoside).

Compound 5 was identified as caffeic acid ethyl ester of the formula (5).

Compound 6 was identified as cinamic acid methyl ester of the formula (6).

Compound 7 was identified as lucidone of formula 7.

Compound 8 was identified as methyl linderone of the formula (8).

Compound 9 was identified as kanakugiol of the formula (9).

Experimental Example  1: the present invention tree extract and Fraction  Antioxidant Activity Measurement

1) 1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging experiment

Electron donating ability was measured according to the DPPH free free radical scavenging method by the Blosis method. That is, 100 μl of various concentrations of samples dissolved in ethanol were dispensed into 96 well plates, and the same amount of 0.4 mM DPPH solution was added thereto, and the absorbance was measured at 517 nm after 10 minutes at room temperature. Butyl hydroxy anisole (BHA) was used as a positive control. DPPH free free radical scavenging activity was calculated from Equation 1 below and expressed as the concentration of the sample (IC ₅₀ ) that appears when the absorbance of DPPH was reduced by 50%.

DPPH free free radical scavenging activity (%) = (A _control -A _sample ) / A _control × 100

Where

A _sample = Absorbance of the reaction solution to which the sample is added,

A _control = Absorbance of the reaction solution to which ethanol was added instead of the sample.

The results for the antioxidant activity of the ethanol extracts and sequential fractions of the birch tree are shown in Table 2 below. The free free radical scavenging activity of DPPH was increased depending on the treatment concentration in both ethanol extract and sequential fractions. And has a DPPH of the free radical scavenging showed a control of higher activity than BHA of active remaining fractions except methylene chloride and hexane fraction also significantly good activity in order ethyl acetate fraction of the fraction, IC ₅₀ value for this time are respectively 7.43 ( EtOAc), 16.88 (EtOH), 18.54 (BuOH), 66.77 μg / mL (H ₂ O) (Table 2).

2) Xanthine oxidase inhibition and superoxide scavenging activity measurement

The uric acid production by xanthine oxidase was measured by the increased absorbance at 290 nm, and allopurinol (Sigma) was used as a control. The amount of superoxide was measured at 560 nm by nitroblue tetrazolium (NBT) reduction. The reaction solution was prepared with various concentrations of 0.5 mM xanthine and 1 mM EDTA in 100 μl of 200 mM phosphate buffer (pH 7.5), and 50 mU / ml xanthine oxidase was added to induce the production of uric acid. Superoxide scavenging activity was reacted by adding 0.5 mM NBT to the reaction solution. Xanthine oxidase inhibition and superoxide scavenging activity were expressed by the concentration of the sample (IC ₅₀ ), which was observed when the absorbance of the produced uric acid and superoxide decreased by 50%, respectively.

The results of xanthine oxidase inhibitory activity and superoxide radical scavenging activity of ethanol extracts and sequential fractions of birch tree are also shown in Table 2. Both the ethanol extract and the sequential fractions of the birch tree showed the concentration-dependent xanthine oxidase inhibitory activity, and the hexane fraction (> 1000ug / ml) and methyllan chloride (IC _50). 86.21 ㎍ / mL) showed slightly lower activity, but IC ₅₀ of other fractions The value was 5.55 ~ 9.79 ㎍ / ㎖ showed a high inhibitory activity (Table 2). In addition, IC ₅₀ in superoxide radical scavenging activity Values were 16.86 μg / ml (EtOAc), respectively, indicating slightly lower activity compared to control allopurinol (IC ₅₀ = 3.82 μg / ml) (Table 2).

This effect, as previously reported, serves to prevent lipid oxidation, oxidative oxygen scavenging and oxidative stress, thereby preventing or delaying aging, cancer and heart disease. Is taking advantage of these effects. Therefore, the birch tree is considered as a useful resource plant for the prevention of many oxidative stresses and damages in vivo when judged based on the antioxidant effect.

Treatment IC ₅₀ (μg / ml) ¹⁾ DPPH radical
scavenging
acivity Uric acid
generation
activity Superoxide
generation
activity EtOH Extract 16.88 ± 0.20 5.55 ± 0.12 63.51 ± 0.01 EtOAc fraction 7.43 ± 0.73 5.56 ± 2.29 16.86 ± 0.09 CH ₂ Cl ₂ Fraction 410.47 ± 10.87 86.21 ± 4.68 806.74 ± 17.14 n -Hex fraction > 1000 > 1000 > 1000 n- BuOH fraction 18.54 ± 2.01 5.66 ± 0.18 58.54 ± 4.01 Water fraction 66.77 ± 2.01 9.79 ± 0.33 270.76 ± 3.93 BHA ^{2 )} 7.47 ± 0.07 N / A N / A Allopurinol N / A 2.09 ± 0.03 3.82 ± 1.06 NOTE ¹ IC ₅₀ values are calculated through regression lines using different concentrations in three replicates.
²⁾ Butylated hydroxyl anisole
³⁾ N / A: Not assay

Experimental Example  2: the present invention tree extract and Fraction MTT assay Cytotoxicity measurement

B16F10 mouse melanoma cells used in this experiment were distributed from the American Cell Line Bank (ATCC). B16F10 cells were treated with DMEM medium containing 10% fetal bovine serum (FBS, Gibco), 1% Antibiotic-Antimycotic (Gibco), L-glutamine and sodium bicarbonate (Gibco) at 37 ° C, 5% CO ₂ Cultured in a cell culture phase. In addition, RAW 264.7 cells were obtained from KCLB (Korean Cell Line Bank) as a murine macrophage cell line using DMEM medium (Gibco) containing 10% FBS, 100 unit / ml penicillin, and 100 ㎍ / ml streptomycin. % CO ₂ Cultured in a cell culture phase.

B16F10 cells were seeded in 24 well plates at 2 × 10 ⁴ cells per well, and at 37 ° C., 5% CO ₂ for 24 hours. After incubation in the cell culture, the samples were incubated for 72 hours by treating the wells at concentrations of 12.5 μg / ml, 25 μg / ml, 50 μg / ml and 100 μg / ml. 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).

Trees ( Lindera erythrocarpa ) Ethanol extracts and sequential fractions were treated and cultured for 3 days at various concentrations up to 12.5 ㎍ / ml, 25 ㎍ / ml, 50 ㎍ / ml and 100 ㎍ / ml to determine the cell viability of B16F10 cell line. Later, the survival rate of the cells was observed by the MTT method. As shown in FIG. 2, when the cell viability of the control group was 100%, the 81-103% at 12.5, 25, 50, and 100 µg / ml concentrations of the ethanol extract and the respective sequential fraction concentrations of the birch ethanol extract were compared to the control group. Slightly decreased or increased. As compared with the control group, the concentrations of 12.5 μg / ml, 25 μg / ml, 50 μg / ml and 100 μg / ml were slightly different but did not show any significant changes. Extracts and sequential fractions are believed to match.

Experimental Example  3: the present invention tree extract and Fraction  Measurement of melanin production inhibitory activity

Inoculate 2 × 10 ⁴ B16F10 cells per well into a 24 well plate and 37 ° C., 5% CO ₂ for 24 hours. After culturing in a cell culture medium, the samples were treated at concentrations of 12.5 ㎍ / ml, 25 ㎍ / ml, 50 ㎍ / ml and 100 ㎍ / ml in each well, and then the medium was removed after 3 days of treatment. Washed twice. 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).

After treatment with ethanol extract and sequential fractions of the non-wood ethanol extract observed to be non-cytotoxic in Experimental Example 2 at various concentrations up to 12.5 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml for 3 days, Melanogenesis inhibitory activity was measured. As shown in FIG. 3, as a control, Arbutin 100 μg / ml, a synthetic substance known as a whitening agent, exhibited 31% melanogenesis inhibitory activity, and 12.5 μg / ml, 25 μg / ml, 50 μg / ml and 100 μg, respectively. The ethanol extract of birch tree showed 0.6%, 7.9%, and 31.6% 45.8% inhibitory activity when treated up to / ml. In addition, at 100 ㎍ / ㎖ concentration of each sequential fraction of 33%, 49%, 24%, 31%, 28% showed a higher melanin inhibitory activity than the control, and showed excellent results as a whitening agent in natural plants. Therefore, these results suggest that the ethanol extract and sequential fractions of the birch tree meet the criteria as a whitening agent that should reduce melanin production and lower cytotoxicity.

Experimental Example  4: the present invention tree extract and Fraction Tyrosinase  Measurement of inhibitory activity

Inoculate 2 × 10 ⁴ B16F10 cells per well into a 24 well plate and 37 ° C., 5% CO ₂ for 24 hours. After culturing in a cell culture medium, the samples were treated at concentrations of 12.5 ㎍ / ml, 25 ㎍ / ml, 50 ㎍ / ml and 100 ㎍ / ml in each well, and then the medium was removed after 3 days of treatment. After washing several times, the cells of the wells were centrifuged to make a cell precipitate, and lysis buffer (0.1 M sodinm phosphate buffer, 0.2 mM PMSF, 1% Triton X-100) was added. After standing on ice to destroy cells and centrifugation, the supernatant was taken and used for enzyme activity measurement. Each sample was added to the reaction solution (12.5 mM L-Dopa, 1.5 mM L-Tyrosine, 67 mM sodium phosphate buffer (pH6.8)) and reacted at 37 ° C for 1 hour using an ELISA reader (μQuant, USA). Absorbance was measured at 405 nm.

Inhibition of the final melanin after treatment with ethanol extract and sequential fractions of birch tree is related to the activity of enzymes involved in melanin synthesis. Therefore, tyrosinase activity in B16F10 cells was 12.5 ㎍ / mL, 25 ㎍ / mL, 50 ㎍ After 3 days of treatment at various concentrations at / ml and 100 ㎍ / mL, Arbutin showed 19% tyrosinase inhibitory activity when treated with Arbutin as a control, and 12.5 ㎍ / mL of ethanol extract and sequential fractions, respectively. , 25 ㎍ / ㎖, 50 ㎍ / ㎖, 100 ㎍ / ㎖ concentration was 13.9%, 15.9%, 36.8%, 42.5% ethanol extract showed a high inhibitory activity. In addition, each fraction also showed higher tyrosinase inhibitory activity than the control group, showing 40%, 21%, 19%, 41%, and 26% at 100 ㎍ / mL. These results suggest that the reduction of melanin production by ethanol extract and sequential fractions of B16F10 cell line may be due to tyrosinase inhibitory activity.

Experimental Example  5: derived from the present invention tree Cyclopentadione  Compound MTT assay Cytotoxicity measurement

1) Lucidone (Compound 1)

In order to determine the effect on the cell viability of B16F10 cell line using a single substance separated by Lucidone, treatment and culture for 3 days at various concentrations up to 1.25 ㎍ / ml, 2.5 ㎍ / ml, 5 ㎍ / ml and 10 ㎍ / ml Afterwards, the survival rate of the cells was observed by MTT method. As shown in FIG. 29, when the cell survival rate of the control group was 100%, the concentration of 1.25, 2.5, 5, and 10 μg / ml of caffeic acid was almost 96-106%, which was slightly decreased or increased compared to the control group. As such, the concentrations of 1.25 μg / ml, 2.5 μg / ml, 5 μg / ml and 10 μg / ml were slightly different but did not show any significant changes, so they were separated into lucidone as a whitening agent that should be low in cytotoxicity. It is believed that a single substance is matched.

2) Methyllinderone (Compound 2)

To examine the effect on the cell viability of B16F10 cell line using single substance separated by methyllinderone, treatment and culture at various concentrations up to 1.25 ㎍ / ml, 2.5 ㎍ / mL, 5 ㎍ / mL and 10 ㎍ / mL for 3 days Afterwards, the survival rate of the cells was observed by MTT method. As shown in FIG. 30, when the cell survival rate of the control group was 100%, the concentration of 1.25, 2.5, 5, and 10 ㎍ / ml of caffeic acid was slightly decreased to 93 to 99% compared to the control group. As compared with the control group, the concentrations of 1.25 ㎍ / mL, 2.5 ㎍ / mL, 5 ㎍ / mL and 10 ㎍ / mL were slightly different but did not show any significant changes, so they were separated into Methyllinderone as a whitening agent that should be low in cytotoxicity. It is believed that a single substance is matched.

3) Kanakugiol (Compound 3)

To examine the effect on the cell viability of B16F10 cell lines using Kanakugiol-separated single substance, treatment and culture at various concentrations up to 1.25 μg / ml, 2.5 μg / ml, 5 μg / ml and 10 μg / ml for 3 days Afterwards, the survival rate of the cells was observed by MTT method. As shown in FIG. 31, when the cell survival rate of the control group was 100%, the concentration of 1.25, 2.5, 5, and 10 ㎍ / ml of caffeic acid was decreased by 91 to 96% compared to the control group. As such, the concentrations of 1.25 μg / ml, 2.5 μg / ml, 5 μg / ml and 10 μg / ml were slightly different, but did not show any significant changes, so they were separated into kanakugiol as the whitening agent that should have low cytotoxicity. It is believed that a single substance is matched.

Experimental Example  6: derived from the present invention tree Cyclopentadione  Determination of Melanin Inhibitory Effects of Compounds

1) Lucidone (Compound 1)

In order to determine the melanogenesis inhibitory effect on B16F10 cell line using a single substance separated by Lucidone, treatment was carried out at various concentrations up to 1.25 ㎍ / ml, 2.5 ㎍ / ml, 5 ㎍ / ml and 10 ㎍ / ml for 3 days, respectively. After melanin production inhibitory activity was measured.

As a result, as shown in FIG. 32, 23.5% of melanin inhibitory activity was observed when 50 μg / ml of arbutin, known as a whitening agent, was treated as 1.25 μg / ml, 2.5 μg / ml, 5 μg / ml, and 10 μg, respectively. Lucidone showed high inhibitory activity of 3%, 19%, 37%, and 53% when treated up to / mL. Therefore, it showed higher melanin inhibitory activity than the control group arbutin and showed good results as a whitening agent in natural plants.

2) Methyllinderone (Compound 2)

To measure the effects of melanin production on the B16F10 cell line using a single substance separated by methyllinderone, treatment was carried out at various concentrations up to 1.25 ㎍ / mL, 2.5 ㎍ / mL, 5 ㎍ / mL and 10 ㎍ / mL for 3 days, respectively. After melanin production inhibitory activity was measured.

As a result, as shown in FIG. 33, 23.5% of melanin inhibitory activity was observed when 50 μg / ml of arbutin, known as a whitening agent, was treated as 1.25 μg / ml, 2.5 μg / ml, 5 μg / ml, and 10 μg, respectively. Methyllinderone showed high inhibitory activity of 1%, 14%, 38%, and 62% when treated up to / mL. Therefore, it showed higher melanin inhibitory activity than the control group arbutin and showed good results as a whitening agent in natural plants.

3) Kanakugiol (Compound 3)

In order to determine the melanin production inhibitory effect on B16F10 cell line using Kanakugiol isolated single substance, it was treated for 3 days at various concentrations up to 1.25 μg / ml, 2.5 μg / ml, 5 μg / ml and 10 μg / ml, respectively. After melanin production inhibitory activity was measured.

As a result, as shown in FIG. 34, 23.5% of melanin production was inhibited by arbutin 50 ㎍ / ml, which is known as a whitening agent, and 1.25 ㎍ / mL, 2.5 ㎍ / mL, 5 ㎍ / mL and 10 ㎍, respectively. Kanakugiol showed high inhibitory activity of 2%, 16%, 29%, and 61% when treated up to / mL. Therefore, it showed higher melanin inhibitory activity than the control group arbutin and showed good results as a whitening agent in natural plants.

Experimental Example  7: derived from the present invention tree Cyclopentadione  Compound Tyrosinase  Inhibition Active measurement

Tyrosinase activity in B16F10 cells was treated with various concentrations of 1.25 ㎍ / ml, 2.5 ㎍ / ml, 5 ㎍ / ml, and 10 ㎍ / ml for 3 days, respectively. The inhibitory activity was higher than that of the control at 10%, 17%, 26.4%, and 48.9% when lucidone was treated with 1.25 ㎍ / ml, 2.5 ㎍ / ml, 5 ㎍ / ml and 10 ㎍ / ml, respectively. Seemed. These results suggest that the reduction of melanin production by lucidone mono-compound isolated from birch tree in B16F10 cell line may be due to tyrosinase inhibitory activity.

As described above in the above Examples and Experimental Examples, the composition for skin whitening containing the extract, fraction or compound of the present invention is effective for extracting or removing the extract from the tree, the solvent fraction thereof or the cyclopentadione compound therefrom. By containing it as an ingredient, it has excellent melanin production inhibitory activity and tyrosinase inhibitory activity, and has a very excellent effect of providing a composition for skin whitening showing higher melanin inhibitory activity and tyrosinase inhibitory activity than Arbutin, which is known as a whitening agent. It is a very useful invention in the cosmetic, pharmaceutical and food industries.

1 is a flow chart briefly illustrating a process of obtaining an ethanol extract and a solvent fraction thereof from a birch tree according to the present invention.

Figure 2 shows the HPLC analysis of the ethanol extract of the birch tree of the present invention.

Figure 3 shows the HPLC analysis of the n -hexane fraction of the ethanol extract of the birch tree of the present invention.

Figure 4 shows the HPLC analysis of the CH ₂ Cl ₂ fraction of the birch ethanol extract of the present invention.

Figure 5 shows the HPLC analysis of the ethyl acetate fraction of the ethanol extract of the birch tree of the present invention.

Figure 6 shows the HPLC analysis of the n -butanol fraction of the birch ethanol extract of the present invention.

Figure 7 shows the HPLC analysis of the water fraction of the ethanol extract of the birch tree of the present invention.

8 is a high performance liquid chromatography result for quantitative analysis of compound 1.

9 shows the ¹ H, ¹³ C-NMR spectrum of Compound 1. FIG.

10 is a high performance liquid chromatography result for quantitative analysis of compound 2.

11 shows the ¹ H, ¹³ C-NMR spectrum of Compound 2. FIG.

12 is a high performance liquid chromatography result for quantitative analysis of compound 3.

13 shows the ¹ H, ¹³ C-NMR spectrum of Compound 3. FIG.

FIG. 14 is a high performance liquid chromatography result for quantitative analysis of compound 4.

15 shows the ¹ H, ¹³ C-NMR spectrum of Compound 4. FIG.

FIG. 16 is a high performance liquid chromatography result for quantitative analysis of compound 5.

17 shows the ¹ H, ¹³ C-NMR spectrum of Compound 5. FIG.

FIG. 18 is a high performance liquid chromatography result for quantitative analysis of compound 6.

19 shows the ¹ H, ¹³ C-NMR spectrum of Compound 6. FIG.

20 is a high performance liquid chromatography result for quantitative analysis of compound 7.

FIG. 21 is a ¹ H, ¹³ C-NMR spectrum of Compound 7. FIG.

FIG. 22 is a high performance liquid chromatography result for quantitative analysis of compound 8.

23 shows the ¹ H, ¹³ C-NMR spectrum of Compound 8. FIG.

FIG. 24 is a high performance liquid chromatography result for quantitative analysis of compound 9.

FIG. 25 is a ¹ H, ¹³ C-NMR spectrum of Compound 9.

FIG. 26 is a graph showing cell viability indicated by ethanol extracts of ethanol and its solvent fractions in B16F10 cells. FIG. The data are mean ± SD values obtained after three measurements.

FIG. 27 is a graph showing melanogenesis inhibitory activity exhibited by ethanol extracts of birch tree and its solvent fractions in B16F10 cells. FIG. The data are mean ± SD values obtained after three measurements.

FIG. 28 is a graph showing the cell tyrosinase inhibitory activity of the ethanol extract and the solvent fraction thereof from B16F10 cells. FIG. The data are mean ± SD values obtained after three measurements.

29 is a graph showing cell viability represented by lucidone in B16F10 cells. The data are mean ± SD values obtained after three measurements.

Fig. 30 is a graph showing cell viability represented by methylinderone in B16F10 cells. The data are mean ± SD values obtained after three measurements.

Fig. 31 is a graph showing cell viability represented by kanakuziol in B16F10 cells. The data are mean ± SD values obtained after three measurements.

32 is a graph showing melanogenesis inhibitory activity exhibited by lucidone in B16F10 cells. The data are mean ± SD values obtained after three measurements.

Fig. 33 is a graph showing melanin production inhibitory activity exhibited by methylinderone in B16F10 cells. The data are mean ± SD values obtained after three measurements.

Fig. 34 is a graph showing melanogenesis inhibitory activity exhibited by kanakuziol in B16F10 cells. The data are mean ± SD values obtained after three measurements.

35 is a graph showing tyrosinase inhibitory activity exhibited by lucidone in B16F10 cells. The data are mean ± SD values obtained after three measurements.

Claims (9)

Lindera erythrocarpa ) Skin whitening composition containing an extract or a solvent fraction thereof as an active ingredient. The composition for skin whitening according to claim 1, wherein the birch tree extract is an ethanol extract. The composition of claim 1, wherein the solvent fraction is a hexane fraction, a methylene chloride fraction, an ethyl acetate fraction, a butanol fraction, a water fraction, or a combination thereof. delete  A composition for skin whitening containing at least one cyclopentadione compound selected from the group consisting of lucidone, methyl linderone and kanakugiol as an active ingredient. The composition for skin whitening according to claim 5, wherein the cyclpentadione compound is separated and purified from a birch tree. The composition for skin whitening according to any one of claims 1 to 3, 5 and 6, wherein the composition for skin whitening is a cosmetic composition. The composition for skin whitening according to any one of claims 1 to 3, 5 and 6, wherein the composition for skin whitening is a pharmaceutical composition. The composition for skin whitening according to any one of claims 1 to 3, 5 and 6, wherein the composition for skin whitening is a food composition.

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