KR20110083089A - Skin whitening composition comprising angelica koreana maxim extract or bisabolangelone compound as active ingredient - Google Patents

Abstract

PURPOSE: A composition containing Angelica koreana Maxim extract or bisabolangelone compound is provided to suppress tyrosinase generation and to develop skin whitening cosmetic product or food. CONSTITUTION: A composition for skin whitening contains Angelica koreana Maxim extract as an active ingredient. The Angelica koreana Maxim extract is isolated using water, anhydrous or hydrous low alcohol of C1-4, acetone, ethyl acetate, or butylene glycol. The composition is a pharmaceutical composition of functional food composition.

Description

Skin Whitening Composition Comprising Angelica koreana Maxim Extract or Bisabolangelone Compound As Active Ingredient}

The present invention is active ( Angelica koreana Maxim) relates to a composition for skin whitening comprising an extract or bisabolangelone (bisabolangelone) compound as an active ingredient.

Melanin pigments are synthesized in the melanocytes of melanocytes through a process called melanogenesis and dispersed into keratinocytes of the epithelium, forming the color of the skin and the skin from the ultraviolet light of the sun. Protect [1]. Biochemical pathways for melanin biosynthesis include two characteristic reactions catalyzed by tyrosinase, the oxidation of L-tyrosine and the oxidation of 3,4-dihydroxyphenylalanine (dopa). Through [2]. The resulting dopaquinone readily reacts with thiol substances such as cysteine or glutathione in the melanosomes to produce yellow / red pheomelanin [3]. When thiol material is depleted, excess dopaquinone spontaneously converts to dopachcrome and eventually forms black / brown eumelanin [3]. Although skin color is determined by the mixing of melanin pigments, tyrosinase is essential for the biosynthesis of both pimelanin and eumelanin.

Paracrine factor secreted from keratinocytes and fibroblasts of the skin can affect the production of melanin in melanocytes. Among these factors, α-MSL (α-melanocyte stimulating hormone) specifically binds to the melanocortin type 1 receptor (MC1R) on melanocytes and activates adenylate cyclase to increase the intracellular concentration of cAMP. Increase, thereby inducing tyrosinase expression [4]. Thus, the balance between paracrine factors plays a very important role in regulating skin pigmentation through fine control of melanocyte activation. However, abnormal accumulation of melanin pigments leads to hyperpigmentation disorders such as melasma, freckles and senile lentigens, which cause arbutin to inhibit tyrosinase activity. Or by treatment with hydroquinone [5].

The inventors of the present invention have conventionally studied to develop pigmentation inhibitors from medicinal plants, and as a result, oxyresveratrol from Mulberry alba, Piperlonguminine and Piperlonguminine from Piper longum Torilin and the like have been isolated from Torilis japonica, and these substances have already been reported to have hypopigmentation [6, 7]. On the other hand, Angelica koreana Maxim is a perennial herb belonging to the genus Ulmliferae, which has been traditionally used in the treatment of headache, neuralgia and joint pain in Korea, but has hypopigmentation effect that inhibits the formation of melanin pigment. Has not been reported to date.

The present inventors have studied to develop melanin pigmentation inhibitors from medicinal plants. As a result, the extract of Angelica koreana Maxim, which has been traditionally used for the treatment of headache, neuralgia, arthralgia, etc., has been used to extract B16 melanoma cells or melanin. -a inhibits the production of melanin induced by α-MSH in the cells, and experimentally indicates that the active ingredient of hypopigmentation in this extract is a bisbobolangelone, a kind of sesquiterpene derivative compound. Confirmed to complete the present invention.

Accordingly, it is an object of the present invention to provide a composition for skin whitening comprising an extract of Angelica koreana Maxim as an active ingredient.

Another object of the present invention is to provide a composition for skin whitening comprising a bisabobolone compound (bisabolangelone) as an active ingredient.

The objects and advantages of the invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention provides a support for Angelica koreana Maxim) provides a composition for skin whitening comprising the extract as an active ingredient.

According to another aspect of the present invention, the present invention provides a composition for skin whitening comprising a bisabolangelon represented by the following formula (1) as an active ingredient.

According to a preferred embodiment of the present invention, the active extract is water, anhydrous or hydrous lower alcohol having 1-4 carbon atoms, acetone, ethyl acetate, butyl acetate, dichloromethane (CH ₂ Cl ₂ ), chloroform, hexane (Hexane) and It provides a composition for skin whitening, characterized in that the solvent extract selected from the group consisting of 1,3-butylene glycol.

According to another preferred embodiment of the present invention, the active extract provides a skin whitening composition, characterized in that it is included in an amount of 0.2-2% by weight.

According to another preferred embodiment of the present invention, the bisabolangelon provides a composition for skin whitening, characterized in that contained in an amount of 0.05-1% by weight.

According to another preferred embodiment of the present invention, the bisabolangelon is active ( Angelica koreana Maxim ) provides a composition for skin whitening, characterized in that separated from the dichloromethane (CH ₂ Cl ₂ ) fraction extract.

According to a preferred embodiment of the present invention, the composition provides a composition for skin whitening, characterized in that the cosmetic composition.

According to another preferred embodiment of the present invention, the composition provides a skin whitening composition, characterized in that the functional food composition.

According to another preferred embodiment of the present invention, the composition provides a skin whitening composition, characterized in that the pharmaceutical composition for the treatment or prevention of melanin hyperpigmentation disease.

According to another preferred embodiment of the present invention, the melanin hyperpigmentation disease provides a skin whitening composition, characterized in that the blemishes, freckles, senile plaques, or solar lentigines.

The active extract in the present invention can be separated according to a conventional method known in the art for extracting the extract from natural products, that is, using a conventional solvent in the presence of conventional temperature, pressure. The active extract in the present invention includes an extract obtained from all parts of the active plant, preferably an extract of leaves, stems or roots, and most preferably an extract of active roots.

As an extraction solvent for extracting the active extract of the present invention, a solvent that can be generally used in the extraction process may be used, and two or more different solvents may be sequentially used for extraction. Preferably, the extractant of the present invention is water, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol), acetone, ethyl acetate, butyl acetate, dichloromethane (CH ₂ Cl ₂ ), chloroform , Hexane (Hexane) and a solvent selected from the group consisting of 1,3-butylene glycol can be used, more preferably methanol, n-hexane, dichloromethane, ethyl acetate, or water, most preferably Methanol or dichloromethane.

The active extract of the present invention is meant to include a fraction further separated by further purification of the extract extracted by the solvent. The further separation is preferably carried out by column chromatography, more preferably size-exclusion chromatography, ion-exchange chromatography, partition chromatography, affinity using liquid column chromatography. It can be separated and purified using (affinity) chromatography or a combination of these chromatography, but is not limited thereto.

Preferably, the active extract of the present invention is a methanol extract, or an extract obtained by using dichloromethane as a solvent in addition to the dichloromethane extract or methanol extract. More preferably, the active extract of the present invention is a fraction extract obtained as a fraction by performing gel filtration chromatography or gel adsorption chromatography on an extract obtained by using dichloromethane as a solvent with respect to the methanol extract. to be.

Another active ingredient of the skin whitening composition of the present invention is a bisabobolangelone compound represented by the formula (1). The bisabolangelon compound is a compound having a sesquiterpene structure.

It is apparent to those skilled in the art that the bisabolangelon compound represented by the formula (1) in the present invention is not only separated from the active extract, but also separated from other sources and chemically synthesized compounds have equivalent skin whitening activity.

In the present invention, the bisabolangelon single compound of Formula 1 from the active extract can be obtained by a conventional method of separating and purifying the compound from the extract. For example, filtration using silica gel or celite gel column, size-exclusion chromatography using ion column chromatography, ion-exchange chromatography, partition chromatography, affinity ( affinity chromatography or a combination of these chromatography can be used for separation and purification.

Single compounds isolated from natural product extracts include Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectroscopy (MALDI-TOF MS), Electron Ionization Mass Spectroscopy (EI MS), Chemical Ionization Mass Spectroscopy (CI), and Fast Atom Bombardment Ionization (FABI). ), Mass spectrometry such as ESI-MS (Electrospray Ionization Mass Spectrometry), and infrared spectroscopy (IR Sectrum), nuclear magnetic resonance (NMR), circular dichroism (CD), etc. have.

The active extract of the composition of the present invention or the bisabolangelon compound of the formula (1) greatly inhibits the level of tyrosinase protein, which is a key enzyme for the synthesis of melanin in the cell, but does not affect the activity of this enzyme. Therefore, the active extract and bisabolangelon of the present invention is a natural source, so it can be used for skin whitening to inhibit the production of melanin without side effects.

The skin whitening composition of the present invention can be used in the form of a cosmetic composition.

Cosmetic compositions of the present invention can be prepared in any formulations conventionally prepared in the art and include, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing It may be formulated as cleansing, oil, powder foundation, emulsion foundation, wax foundation, spray, and the like, but is not limited thereto. More specifically, it may be prepared in the form of a flexible lotion, nutrition lotion, 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, lotion, or gel, the carrier component is animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide. This can be used.

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.

Ingredients included in the cosmetic composition of the present invention, in addition to the active ingredient and the carrier component, include ingredients conventionally used in cosmetic compositions, for example, such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavorings Adjuvants may be included.

The skin whitening composition of the present invention can be used in the form of a functional food composition.

Functional food compositions of the present invention include ingredients that are commonly added in the manufacture of food, and include, for example, proteins, carbohydrates, fats, nutrients and seasonings. For example, when prepared with a drink, flavoring agents or natural carbohydrates may be included as additional ingredients in addition to the active ingredient. For example, natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.); Disaccharides (eg maltose, sucrose, etc.); oligosaccharide; Polysaccharides (eg, dextrins, cyclodextrins, etc.); And sugar alcohols (eg, xylitol, sorbitol, erythritol, and the like). As the flavoring agent, natural flavoring agents (e.g., taumartin, stevia extract, etc.) and synthetic flavoring agents (e.g., saccharin, aspartame, etc.) can be used.

The skin whitening composition of the present invention can be used in the form of a pharmaceutical composition for the treatment or prophylaxis of melanin hyperpigmentation disease.

As used herein, the term “hyperpigmentation of melanin pigment” means blacking or darkening in comparison to other areas due to excessive increase of melanin in certain areas of the skin or nails. Preferably, the melanin hyperpigmentation disease includes, but is not limited to, blemishes, freckles, senile plaques, solar lentigines, and the like.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier in addition to the active ingredient, and such carriers are commonly used in the preparation of 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 oils and the like, but is not limited thereto. 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. Suitable pharmaceutically acceptable carriers and formulations are Remington's Pharmaceutical Sciences (19th ed., 1995).

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. On the other hand, the oral dosage of the pharmaceutical composition of the present invention is preferably 0.0001-100 mg / kg (body weight) per day.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and when administered parenterally, may be administered topically to the skin, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, and the like. . In view of the fact that the pharmaceutical composition of the present invention is applied for the treatment or prevention of melanin hyperpigmentation disease, the administration of the composition of the present invention is preferably applied topically to the skin.

The concentration of the active ingredient included in the composition of the present invention can be determined in consideration of the purpose of treatment, the condition of the patient, the period of time, etc., and is not limited to a specific range of concentration.

The pharmaceutical compositions of the present invention may be prepared in unit dosage 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 incorporating into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or an aqueous medium, or may be in the form of extracts, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer.

According to a preferred embodiment of the present invention, the pharmaceutical composition of the present invention has an external dermal formulation. The external dermal formulation is not particularly limited and is preferably a powder, gel, ointment, cream, lotion, liquid or aerosol formulation.

The present invention relates to a composition for use in skin whitening comprising an active extract or a bisabolangelon compound as an active ingredient. The active extract or bisabolangelon compound of the present invention significantly inhibits the production of tyrosinase, which is a key enzyme of melanin synthesis, and is extracted from natural products and has almost no side effects to the human body. It can be developed as a medicinal or functional food.

1 is a view showing a process for fractionating the active extract using a solvent or column chromatography in the present invention.
Figure 2 shows the IR spectral results of bisabolangelone (bisabolangelone).
Figure 3 shows the ESI-MS spectral results of bisabolangeloron.
4 shows the ¹ H-NMR spectral results of bisabolangelolon.
5 shows the ¹³ C-NMR spectral results of bisabolangelones.
Figure 6 shows the chemical structure of the bisabolangelon compound of the present invention.
7A-7D show the effect of bisabolangelolon on melanin production. B16 cells were stimulated with α-MSH for 72 hours in the presence of bisabolangelon or arbutin. The amount of melanin pigment in the cells was measured using synthetic melanin as a standard (FIG. 7A), and the effect of bisabolangelon (-●-) or arbutin (-○-) was expressed as percent inhibition (FIG. 7B). Melan-a cells were stimulated with α-MSH for 96 hours in the presence of bisabolangelon and quantitated melanin pigments intracellularly (FIG. 7C). B16 cells were incubated for 72 hours with various concentrations of bisabolangelon in the presence of α-MSH. Viable cells were measured by the MTT method (FIG. 7D). Data are expressed as mean ± SD values for 3-5 independent experiments. ^# P <0.05 vs. Medium only group. ^* P <0.05 vs. α-MSH alone stimulation group.
Figures 8a and 8b is a result of measuring the effect of the bisabolangelon compound of the present invention on tyrosinase protein expression and activity. B16 cells were stimulated with α-MSH for 48 hours in the presence of bisabolangelolone. Cell extracts were subjected to Western blot analysis using anti-tyrosinase or anti-GAPDH antibodies (FIG. 8A). Cell free tyrosinase was prepared from B16 cells (hatched bars) and tyrosinase of mushroom origin was prepared by purchasing a commercially available type (black bars). The enzyme solution was treated with bisabolangelon or arbutin, and then the oxidation rate of L-tyrosine was measured by spectroscopic method as catalytic activity of tyrosinase (FIG. 8B). Tyrosinase 1 unit activity was defined as the conversion of 1 nmole of L-tyrosine per minute to dopachrome. Data are expressed as mean ± SD from five independent experiments. ^* P <0.05 vs. Tyrosinase alone-containing group.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example

Materials and Methods

Example 1. Compounds, Antibodies, and Instruments

Fetal bovine serum (FBS) and the materials used for the culture were purchased from Invitrogen. Arbutin (> 98% purity), synthetic melanin and α-MSH were purchased from Sigma-Aldrich. Antibodies against tyrosinase and antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Santa Cruz Biotech. UV and IR spectral analysis results were obtained from JASCO UV-550 and Perkin-Elmer model LE599 spectrometers, respectively. NMR spectral results were obtained using a Bruker AMX 500 MHz NMR spectrometer. ESI-MS spectra were measured using LCQ Fleet LC / MS (Thermo Scientific Co.).

Example 2. Plant Materials

The dried root of Angelica koreana Maxim. (Umbelliferae) was purchased from the traditional Chinese herbal medicine market in Cheongju and used after botanical identification. Some were deposited with Chungbuk National University as evidence samples (CBNU46).

Example  3. Bioanalysis -Judo( bioassay - guided Isolation of Plant-Derived Compounds

The dry roots of the actives were extracted with 100% methanol at room temperature and the solvent was evaporated under reduced pressure. Methanol extract (640 g) was resuspended in H ₂ O, and fractionated sequentially using n-hexane (hexane), CH ₂ Cl ₂ and ethyl acetate (ethylacetate). Referring to FIG. 1, which illustrates a method for isolating bioassay-guided plant compounds of the present invention, a CH ₂ Cl ₂ fraction (41 g) is further added to a silica gel column (7 × 25 cm, 200-400). After loading on the mesh), n-hexane / acetone gradient eluates (95: 5, 90:10, 80:20, 70:30, 50:50, 0: 100) were each eluted using 2.5 L. . Eluates were separated into 9 fractions (Fr-1 to Fr-9) combined based on TLC pattern. Among these fractions, fraction Fr-3 showed an IC ₅₀ value of 3.2 μg / ml on α-MSH induced melanogenesis in B16 cells, showing the strongest inhibitory effect. From the fraction Fr-3 (14 g), the active ingredient (370 mg) was isolated by recrystallization in 100% methanol and analyzed by spectral data, indicating that the substance was bisabolangelone (purity:> 97%). Confirmed.

Example 4 Bisabolangelone Compounds

Bisabolangelon compounds were observed as crystals of weak yellow needle shape. Spectroscopic data for this compound were as follows: UV (MeOH): λ _max = 247; IR (KBr): ν _max = 3342 (−OH), 1640 (C═O) cm ⁻¹ (FIG. 2); ESI-MS: 249 [M + H] ⁺ (Figure 3); ¹ H-NMR (500 MHz, CDCl ₃ ): δ 6.02 (1H, m, H-5), 6.01 (1H, m, H-11), 5.39 (1H, d, = 11.5 Hz, H-10), 4.89 (1H, q, J = 6.0 Hz, H-8), 2.75 (2H, m, H-7), 2.67 (1H, d, J = 6.5 Hz, H-9), 2.03 (3H, s, 6- CH ₃ ), 1.81 (3H, s, 12-CH ₃ ), 1.74 (3H, s, 12-CH ₃ ), 1.64 (3H, s, 3-CH ₃ ) (FIG. 4); ¹³ C-NMR (125 MHz, CDCl ₃ ): δ 196.8 (C-4), 160.0 (C-6), 158.1 (C-2), 132.4 (C-12), 127.2 (C-5), 117.7 (C -11), 94.3 (C-10), 78.5 (C-3), 76.1 (C-8), 53.6 (C-9), 34.9 (C-7), 27.4 (C-15), 26.0 (C- 13), 24.7 (C-16), 18.0 (C-14) (FIG. 5).

Example  5. Cell culture Cell culture )

B16 melanoma cells and melan-a melanocytes were purchased from the American ype Culture Collection (ATCC). B16 cells were treated with DMEM medium (13.4 mg / ml Dulbecco's modified Eagle's medium, 10 mM HEPES, 24 mM NaHCO3, supplemented with 10% FBS and antibiotic (100 U / ml penicillin G sodium, 100 μg / ml streptomycin sulfate). cultured at pH 7.1), melan-a cells were cultured in RPMI medium (10.4 mg / ml RPMI 1640, 10 mM HEPES, 24 mM NaHCO3) with 10% FBS, 200 nM PMA (phorbol 12-myristate 13-acetate) and antibiotics. , pH 7.1). Both cells were incubated at 37 ° C. under humidified conditions with 5% CO ₂ .

Example  6. Quantification of Melanin Pigments

Cells were stimulated with 10 nM α-MSH in the presence of a sample for 72 hours for B16 cells and 96 hours for melan-a cells. The content of melanin was measured according to a known method [8]. Briefly, cells were harvested and then disrupted by heating to 80 ° C. in 1N NaOH-10% dimethyl sulfoxide. The amount of melanin pigment was determined by measuring absorbance at 405 nm with synthetic melanin (> 95% purity) as standard.

Example  7. Cell Viability  analysis

Cells were incubated with various concentrations of bisabolangelon in the presence of 10 nM α-MSH and MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium bromide] assays were performed. . Briefly, 0.5 mg / ml MTT was added to the cells and incubated for 3 hours. Cell viability was measured by observing the conversion of MTT into purple formazan by active cells with metabolism. Formazan crystals were dissolved in 100% dimethylsulfoxide and then absorbance was measured at 590 nm.

Example  8. Weston Blotting  analysis

Cells are stimulated with 10 nM α-MSH in the presence of a sample for 48 hours, followed by lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM NaF, 1 mM EDTA, 0.1% SDS, 0.5% NP-10 , 1 mM PMSF, 10 mM Na ₃ VO ₄ , pH 7.4). Cell extracts were separated by electrophoresis on SDS-acrylamide gels and transferred to PVDF membranes. Blots of PVDF membranes were incubated overnight at 4 ° C with anti-tyrosinase antibodies or anti-GAPDH antibodies. It was then incubated at room temperature for 2-4 hours with a suitable horseradish peroxidase-conjugated secondary antibody. Finally the blots were reacted with enhanced chemiluminescence (ECL) reagent (GE Healthcare) and then exposed to X-ray film.

Example  9. Tyrosinase Activity Assay

Enzymes were purchased as commercially available mushroom tyrosinase (Sigma-Aldrich) and prepared separately from B16 cells. Cells were stimulated with 10 nM α-MSH for 48 hours and disrupted by ultrasonic grinding on ice. After centrifugation, the supernatant was dialyzed against 50 mM sodium phosphate buffer (pH 6.8) and used as an enzyme source of B16 cell source. Tyrosinase activity was assessed by measuring L-tyrosine oxidation rates according to known methods [6]. Briefly, samples of the present invention for which activity is to be measured were incubated with enzymes derived from B16 cells or mushrooms, and then 5 mM L-tyrosine was reacted as a substrate. The rate of dopachrome formation was determined by the rate of increase in absorbance at 475 nm. One unit of enzymatic activity was defined as conversion of 1 nmole of L-tyrosine per minute to dopachrome.

Example  10. Statistical Analysis

Data are expressed as mean ± SD and analyzed by ANOVA and Dunnet's test. A value of p <0.05 was considered statistically significant.

Experiment result

Example  11. Extract and Active Ingredient Analysis

In the experiment of the present invention, the total methanol extract of A. koreana root inhibited the production of melanin in α-MSH-activated B16 cells (see Table 1).

TABLE 1 Inhibitory activity of solvent extract or column chromatography fraction extract on α-MSH induced melanin production in B16 cells.

In order to elucidate the hypopigmenting principle of the active extract, methanol extract was further fractionated into n-hexane layer, CH ₂ Cl ₂ layer, ethyl acetate (EtOAc) layer and aqueous layer to measure activity. As a result, CH ₂ Cl ₂ The layer showed the best activity (see Table 1). The CH ₂ Cl ₂ layer was further subjected to bioassay-guided fractionation using column chromatography techniques. By separating the active ingredient from the extract and comparing it with the reported spectral data [9], it was confirmed that the compound was a bisabobolangelone compound, which is a sesquiterpene derivative (see FIGS. 2 to 6).

Example  12. Evaluation of whitening activity

B16 cells or melan-a cells were stimulated with α-MSH in the presence or absence of bisabolangelones. The content of melanin in the unstimulated B16 cells was very low at 63 ± 28 pg / cell, but increased significantly to 348 ± 25 pg / cell when stimulated with α-MSH alone for 72 hours (see FIG. 7A). ).

Treatment of bisabolangelones in B16 cells inhibited the production of melanin induced by α-MSH in a dose dependent manner against the nonsabolangelones treated. That is, while IC ₅₀ value had 2 g / mL, it showed 28% inhibition at 1 g / mL, 90% inhibition at 3 g / mL, and 99% inhibition at 10 g / mL (FIGS. 7A and 7B). Reference). Arbutin is a skin lightening agent well known in the art [10]. When arbutin was treated in α-MSH stimulated B16 cells, the IC ₅₀ value was 96 μg / mL and melanogenesis was suppressed (FIG. 7B).

Consistent with the results in B16 cells, 96 hours of incubation with α-MSH even in melan-a cells, 104 ± 8 pg / at baseline 38 ± 3 pg / cell The melanin production was significantly increased up to the level of the cells, and the mesa-a cells stimulated with α-MSH showed mesogenic inhibition of melanin with an IC ₅₀ value of 4 μg / mL (FIG. 7C). On the other hand, because the non-sabolangelon did not affect the viability of B16 cells at the effective concentration showing the hypopigmenting response (hypoigmenting response), the possibility of experimental results by non-specific cytotoxicity could be excluded (Fig. 7d). The experimental results showed that the bisabolangelones of the present invention can reduce melanin production without cell-type specificity.

Example  13. Whitening Activity Mechanism  analysis

Tyrosinase is an enzyme that catalyzes the hydroxylation and dopa oxidation of L-tyrosine, a rate determining step in the biosynthetic pathway of melanin pigments, which induce their expression in the presence of α-MSH. Properties [2, 4]. In order to observe the mechanism by which bisabolangelones inhibit melanin production, it was further investigated whether bisabolangelones influence the amount of tyrosinase in the cells. That is, Western blot analysis of tyrosinase protein was performed after B16 cells were stimulated with α-MSH for 48 hours in the presence or absence of bisabolangelon. As shown in FIG. 8A, the levels of tyrosinase protein increased significantly compared to basal levels when cells were exposed to α-MSH only. However, treatment with bisabolangelon reduced the levels of tyrosinase induced by α-MSH in a dose dependent manner. On the other hand, the expression level of GAPDH that is constantly expressed was not affected by the stimulation with α-MSH or the treatment with bisabolangelolon.

The inventors then investigated whether bisabolangelones directly affect the catalytic activity of tyrosinase. Tyrosinase in cell-free state was prepared from B16 cells, and also commercially available type of tyrosinase of mushroom origin. The solution of the enzyme prepared above was treated with bisabolangella, and the oxidation rate of L-tyrosine was measured as a tyrosinase activity measure. Treatment with bisabolangeloron did not significantly affect the catalytic activity of both B16 cells and tyrosinase isolated from the mushroom, but the positive control arbutin was 59-68% at a concentration of 80 μg / mL. Showed inhibition of tyrosinase activity (FIG. 8B).

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

references

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Claims (12)

Angelica koreana Maxim) Skin whitening composition comprising the extract as an active ingredient.
The method of claim 1, wherein the active extract is water, anhydrous or hydrous lower alcohol having 1-4 carbon atoms, acetone, ethyl acetate, butyl acetate, dichloromethane (CH ₂ Cl ₂ ), chloroform, hexane (Hexane) and 1,3 A solvent extract selected from the group consisting of butylene glycol.
The composition of claim 1 or 2, wherein the active extract is contained in an amount of 0.2-2 wt%.
The composition of claim 1 wherein the composition is a cosmetic composition or a functional food composition.
The composition of claim 1, wherein the composition is a pharmaceutical composition for the treatment or prevention of melanin hyperpigmentation disease.
6. The composition of claim 5, wherein the melanin hyperpigmentation disease is blemishes, freckles, senile plaques, or solar lentigines.
Skin whitening composition comprising a bisabolangelone (bisabolangelone) represented by the formula (1) as an active ingredient.
[Formula 1]

8. The composition of claim 7, wherein the bisabolangelon is included in an amount of 0.05-1 wt%.
The composition of claim 7, wherein the bisabolangelolon is isolated from dichloromethane (CH ₂ Cl ₂ ) fraction extract of Angelica koreana Maxim.
8. The composition of claim 7, wherein the composition is a cosmetic composition or a functional food composition.
According to claim 7, wherein the composition is a composition characterized in that the pharmaceutical composition for the treatment or prevention of melanin hyperpigmentation disease.
12. The composition of claim 11, wherein the melanin hyperpigmentation disease is blemishes, freckles, senile plaques, or solar lentigines.

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