KR101047588B1 - Whitening composition comprising white rice extract and novel pregnan compound isolated therefrom as active ingredient - Google Patents

Abstract

The present invention relates to a whitening cosmetic composition comprising a Cynanchum atratum extract and a novel pregnane-based compound isolated therefrom and a pharmaceutical composition for the treatment or prevention of hyperpigmentation diseases. The white rice extract, fractions and pregnancies of Formula 1 and 2 separated therefrom exhibit excellent whitening activity and thus can be developed as cosmetics for whitening and pharmaceuticals for the treatment or prevention of diseases caused by hyperpigmentation. .

Cynanchum atratum, extract, fraction, whitening, hyperpigmentation disease, tyrosinase, melanin

Description

Composition for Skin Whitening Comprising the Extraction of Cynanchum atratum and Novel Pregnane Derivatives Purified from the Same as an Active Ingredient}

The present invention relates to a whitening cosmetic composition comprising a Cynanchum atratum extract and a novel pregnane-based compound isolated therefrom, and a pharmaceutical composition for the treatment or prevention of hyperpigmentation and deposition disorders. .

White rice ( Cynanchum atratum ) is a perennial herb that grows in the mountain family belonging to the family of gourds. Flowers bloom in May-July, black purple, hang on the end of small peduncles splitting from axilla to ridge. Fruits are peduncles, broad lanceolate, ripen in September-October, 7-8cm long, with dense hairs on the outside, with long white hairs on seeds. The greenish color of flowers is called blue white flowers (for. Viridescems ). Traditionally, herbal medicines have prescribed roots and rhizomes for fever, diuresis, and edema. In the private sector, leaves have been used as tonics.

Korean Patent Publication No. 10-2002-0029181 discloses a whitening cosmetic composition comprising an extract using a solvent such as ethanol, methanol, purified water, acetone, ethyl acetate, and the like of white rice, but whitening activity in white rice extract. There is no disclosure of a whitening effect at the level of a single compound on a bioactive substance that exhibits the following properties.

Melanine, which is a target in the whitening activity experiment, is a polymer of phenols widely present in animals, plants, and microorganisms, and its production is known to form spots, freckles, and aging skin. Melanin is synthesized by the continuous oxidation of tyrosinase enzymes in the melanosomes in the melanocytes of the epidermal basal layer. Tyrosinase is a major enzyme in the melanin biosynthesis process containing copper and the starting material of melanin synthesis is tyrosine, a type of amino acid. Tyrosine is oxidized to L-3,4-dihydroxyl-L-phenylalanine (L-DOPA) by tyrosinase, which in turn is oxidized to DOPAquinone and DOPAquinone to DOPAchrome, 5,6-dihydroxyin-dole, indole 5,6-quinone Then, melanin is finally synthesized by polymerization (1, 2). Tyrosinase is a tyrosine hydroxyase that oxidizes tyrosine to make DOPA. It acts as a DOPA oxidase that oxidizes DOPA to make DOPA quinone and plays an important enzyme in synthesizing melanin polymers (3). Therefore, tyrosinase activity inhibition experiment is recognized as a useful evaluation method. Tyrosinase activity inhibition experiment is classified into tyrosine hydroxylase inhibition test, DOPA oxidase inhibition test, and comprehensive melanin synthesis inhibition test. Since silase inhibitory assay and melanin synthesis inhibitory assay use radioisotopes, DOPA oxidase inhibition assays that measure the amount of DOPAchrome produced using DOPA as a substrate are frequently used (4). . Tyrosinase inhibitors known to date include hydroquinone, resorcinol, 4-hydroxyanisole, ascorbic acid and its derivatives, kojic acid, and uva. There are arbutin, glucosamin, oxyreseveratrol, alpha-viniferin, and ferulic acid, but arbutin and kojic acid value due to problems such as skin safety and formulation stability Only limited amounts of additives for whitening agents are used (5, 6).

Various plants have been studied until recently to develop natural whitening agents. Among them, tyrosinase inhibitory activity is known to be sangbaekpi (7, 8), baejaja (9), licorice (10), green tea (11), Suk ( 12), Gardenia fruit (13) and the like have been reported, some of which are used in the product.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have diligently researched to extract an active ingredient having improved skin whitening effect among various natural products. As a result, the extract of Cynanchum atratum has the inhibitory activity of tyrosinase and the inhibitory activity of melanin production. It is experimentally confirmed that the whitening activity is very excellent through the activity guided fractionation and isolation (activity guided fractionation and isolation) specific white rice fraction and the new pregnane compound separated and purified from this fraction showing the whitening activity The present invention was completed by experimentally identifying that.

Accordingly, it is an object of the present invention to provide a composition for whitening comprising white rice ( Cynanchum atratum ) extract as an active ingredient.

Another object of the present invention is to provide a novel pregnane compound having a whitening activity isolated from the extract of Cynanchum atratum .

Further, another object of the present invention is to provide a whitening composition comprising a novel pregnane compound isolated from the extract of Cynanchum atratum as an active ingredient.

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

According to one aspect of the present invention, the present invention is (a) a cosmetically effective amount of Cynanchum atratum extract; And (b) provides a cosmetic composition for whitening comprising a cosmetically acceptable carrier.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition comprising (a) a pharmaceutically effective amount of Cynanchum atratum extract; And (b) provides a pharmaceutical composition for the treatment or prevention of hyperpigmentation disease comprising a pharmaceutically acceptable carrier.

According to another aspect of the present invention, the present invention provides a novel pregnane-based compound represented by the following Chemical Formula 1 or Chemical Formula 2 separated and purified from white rice extract.

[Formula 1]

[Formula 2]

According to another aspect of the present invention, the present invention is (a) a cosmetically effective amount of a pregnane compound represented by the formula (1) or (2); And (b) provides a cosmetic composition for whitening comprising a cosmetically acceptable carrier.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition comprising (a) a pharmaceutically effective amount of a pregnane compound represented by Formula 1 or Formula 2; And (b) provides a pharmaceutical composition for the treatment or prevention of hyperpigmentation disease comprising a pharmaceutically acceptable carrier.

The present inventors have diligently researched to extract an active ingredient having an improved skin whitening effect among various natural products. As a result, Cynanchum experimentally confirmed that atratum extract has very excellent whitening activity through the inhibitory activity of tyrosinase and the inhibitory activity of melanin production, and the specific white rice fraction by activity guided fractionation and isolation. The present invention was completed by experimentally identifying that the new pregnane compound separated and purified from this fraction is an active ingredient showing whitening activity.

As used herein, the term " Cynanchum atratum extract "means a mixture obtained by extracting from a white rice using a suitable solvent." Fractal fraction "herein refers to extracting the original white rice crude extract back into a specific solvent, The method of separating white rice extract or fractions according to the present invention is performed according to conventional methods known in the art for extracting extracts from natural products, that is, under the conditions of conventional temperature and pressure. Phosphorus solvent can be used to separate.

As an extraction solvent for extracting the white rice extract in the composition of the present invention, a solvent that can be generally used in the process of obtaining an extract from natural products may be used. For example, water, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms , Acetone, ethyl acetate, butyl acetate, 1,3-butylene glycol, hexane, and methylene chloride, but is not limited thereto.

According to a preferred embodiment of the present invention, the extract of Cynanchum atratum which is included as an active ingredient in the cosmetic or pharmaceutical composition of the present invention is a methanol (methanol) solvent extract of white rice.

According to another preferred embodiment of the present invention, the whitening ( Cynanchum atratum ) extract included as an active ingredient in the cosmetic or pharmaceutical composition of the present invention is a whitening active fraction of methylene chloride solvent of the methanol solvent extract of white rice (fraction) )to be.

According to another preferred embodiment of the present invention, the Cynanchum atratum extract included as an active ingredient in the cosmetic or pharmaceutical composition of the present invention is suitable for methylene chloride solvent whitening active fraction of methanol solvent extract of white rice A whitening active fraction obtained through silica gel column chromatography using an elution solvent and / or octadecylsilyl silica column chromatography.

According to another preferred embodiment of the present invention, the elution solvent in the chromatography used to obtain the whitening active fraction is a methylene chloride-methanol solvent or a hexane-acetone solvent.

Pregnane-based compounds represented by Formulas 1 and 2 included as an active ingredient in the composition of the present invention is separated from the Cynanchum atratum extract by performing a conventional separation and purification process. Separation via various purification methods additionally carried out, for example, separation using an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity). do.

According to a specific embodiment of the present invention, the compound of formulas 1 and 2 of the present invention is white rice ( Cynanchum methanol solvent extract of atratum , fractionation using methylene chloride solvent, silica gel column chromatography using methylene chloride-methanol solvent, silica gel column chromatography using hexane-acetone solvent Purified by active tracer separation via ODS column chromatography using methylene chloride-methanol solvent and high performance liquid chromatography (HPLC) using acetonitrile-methanol (CH ₃ CN-MeOH) solvent. It is a whitening compound.

Pregnane-based compounds represented by Formula 1 and Formula 2 purified from the extract of Cynanchum atratum of the present invention is a novel compound first reported in nature.

Since the Fregnan-based compound of Formula 1 and Formula 2 of the present invention has a whitening activity, it can be used as an active ingredient in a cosmetic composition for whitening.

The cosmetic composition for whitening of the present invention may include other whitening components in addition to the Fregnan compound of Formula 1 or Formula 2 as an active ingredient. However, the cosmetic composition of the present invention exhibits a very good whitening effect even with an active ingredient composed only of the compound of formula 1 or formula 2 above.

As used herein, the term "cosmetic effective amount" means an amount sufficient to achieve the skin whitening efficacy of the above-described pregnanci-based compound of the present invention.

The ingredients included in the cosmetic composition of the present invention include ingredients commonly used in cosmetic compositions in addition to the above ingredients as active ingredients, and include, for example, conventional auxiliaries such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavors, and Carrier.

Cosmetic compositions of the present invention may be prepared in any formulation conventionally 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 and sprays, and the like, but are not limited thereto. More specifically, it may be prepared in the form of a flexible lotion (skin), nourishing lotion (milk lotion), nourishing 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 emulsion, a solvent, solubilizer or emulsifier is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 Fatty acid esters of, 3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan.

When the formulation of the present invention is a suspension, a liquid diluent such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, and microcrystals are used as carrier components. 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, methyltaurate, sarcosinate, 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.

Cynanchum atratum extract of the present invention, the active fraction and the pregnane compound represented by the formula (1) and (2) isolated from the pharmaceutical composition for the treatment or prevention of diseases caused by hyperpigmentation (Hyperpigmentation) It can be used as an active ingredient of.

As used herein, the term "disease due to hyperpigmentation" refers to a disease that becomes darker or darker than other areas due to excessive increase of melanin in certain areas of the skin or nails. Preferably, the hyperpigmentation disease includes, but is not limited to, blemishes, freckles or senile plaques, solar lentigines, and the like.

Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those 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 oil, and the like. It is not limited. 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 described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995) .

Suitable dosages of the pharmaceutical compositions of the invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex of the patient, degree of disease symptom, food, time of administration, route of administration, rate of excretion and response to reaction. In general, the skilled practitioner can readily determine and prescribe a dosage effective for the desired treatment. On the other hand, the dosage of the pharmaceutical composition of the present invention is preferably 0.001-2000 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 by intravenous infusion, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, or the like. It is preferable that the route of administration of the pharmaceutical composition of the present invention is determined according to the type of the disease to be applied. For example, the pharmaceutical composition of the present invention is preferably administered in a manner applied topically to the skin because it is used in the treatment of skin diseases caused by overdeposition of melanin pigment.

The pharmaceutical compositions of the invention may be formulated in unit dose form by formulating with pharmaceutically acceptable carriers and / or excipients, according to methods readily available to those of ordinary skill in the art. It can be made or 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.

Cynanchum atratum extracts, fractions and pregnane compounds of Formula 1 and Formula 2 isolated therefrom of the present invention exhibit excellent whitening activity, so as to treat or prevent whitening cosmetics and hyperpigmentation diseases It can be developed as a medicine.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experiment method

1. Measurement of melanin production at the cellular level

Mouse melanoma B16F0 cells suspended in DMEM medium containing 10% FBS were aliquoted in a 96-well plate at 2.5 × 10 ³ cells / well, and treated at 37 ° C., 5% CO ₂ for one day. Incubated. Samples serially diluted with stimulants alpha-MSH (100 nM), forskolin (20 uM), IBMX (100 uM) or db-cAMP (2 mM) were then treated for 3 days. The amount of melanin released into the medium was measured by absorbance at a wavelength of 405 nm. The experiment was repeated three times or more, and the rate of inhibiting melanin production, that is, the whitening activity was expressed as a percentage and an ED ₅₀ value.

2. Tyrosinase and Western Blot Analysis TRP -1 Protein production measurement method

Mouse melanoma B16F0 cells were dispensed in 6-well plates at 5 × 10 ⁴ cells / well and incubated for one day. Subsequently, the samples diluted in series with the stimulant IBMX (100 uM) were treated and further incubated for 48 hours. Cells were collected and then lysis buffer (50 mM Tris, pH 7.4, 0.1% SDS, 1% NP-40, 150 mM NaCl, 1 mM PMSF, 10 mM NaF, 10 ug / ml aprotinin, 10 ug / ml Cell lysate was prepared using leupeptin, 10 mM Na ₃ VO ₄ ). Cell lysates containing the same amount (10-20 ug) of protein were separated by 10% SDS-PAGE, followed by western blotting using a PVDF membrane. Blotting was then reacted with anti-tyrosinase antibody or anti-TRP-1 antibody as a primary antibody at 4 ° C. overnight. The blot was washed three times with TTBS buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween-20) and reacted with a secondary antibody (horseradish peroxidase-conjugated anti-goat IgG) for 2 hours at room temperature. I let you. The blot was again washed three times with TTBS buffer, reacted with an ECL kit in the dark and exposed to X-ray film to identify each protein.

3. Method for measuring enzyme activity of tyrosinase

Melanoma B16F0 cells treated with stimulant IBMX (100 uM) for 2 days were collected and then in lysis buffer (63 mM sodium phosphate buffer, pH 6.8, 0.5% Triton X-100, 0.1 mM PMSF). After suspension, sonication was performed to crush the cells, and the supernatant obtained by centrifugation at 15,000 rpm for 20 minutes was used as an enzyme source. Enzyme reaction was performed by adding 80 ul of 63 mM sodium phosphate buffer (pH 6.8), 40 ul of substrate L-dopa, 40 ul of sample to 40 ul of sample, and then producing dopachrome at 37 ° C. Velocity was measured by absorbance at wavelength 475 nm.

Experiment result

1. Preparation and Activity-Oriented Lineage Fraction of White Rice Extract

(1) Solvent Fraction of White Rice Extract

3 kg of Cynanchum atratum , a plant belonging to Asclepiadaceae, was purchased from Gyeongdong Market, and then chopped and extracted with 15 L of methanol (MeOH) three times at room temperature three times. Methanol (MeOH) extract (300 g) was solvent fractionated using hexane, methylene chloride (MC), ethyl acetate (ethylacetate, EtOAc) and water (H ₂ O).

(2) column fractions for active fractions

As a result of searching for whitening activity through activity analysis, it was confirmed that the fraction having activity was methylene chloride (MC) layer. Thus, methylene chloride (MC) fraction (120 g) was subjected to silica gel column chromatography with methylene chloride-methanol (MC-MeOH) solvent to obtain 10 fractions (CA-MC-FR-1 to CA-MC). -FR-10).

(3) Column fraction for white rice fraction CA-MC-FR-4

The CA-MC-FR-4 fractions with the strongest whitening activity were repeatedly subjected to silica gel column chromatography with hexane-acetone solvent and then separated into 15 small fractions (CA-C4-A). Up to CA-C4-O).

(4) Column fraction for white rice fraction CA-C4-L

The CA-C4-L fraction with the strongest whitening activity was subjected to ODS (octadecylsilyl silica) column chromatography with methylene chloride-methanol (CH ₂ Cl ₂ -MeOH) solvent to obtain 7 small fractions (CA-C4-L21 to CA-). Up to C4-L27).

(5) Separation and purification of active ingredient through preparative HPLC on white rice fraction CA-C4-L25

The CA-C4-L25 fraction with the strongest whitening activity was prep. With CH ₃ CN-MeOH solvent (flow rate 6 mL / min). HPLC was performed to separately separate Compound 1 (20 mg) and Compound 2 (15 mg) having whitening activity.

2. Identification of chemical structure of pure compound

(1) Compound 1

In the NMR spectrum of Compound 1 isolated from the CA-C4-L25 active fraction, six methyl groups were observed in ¹ H NMR, and two olefinic protons at 6.00 and 5.48 ), δ 4.98, 4.84, and 4.77 showed an anomeric proton originating from the three sugars. In addition, a total of 42 carbons were identified in the ¹³ C NMR spectrum, one ketone at δ 212.11, and one ketofuran group at δ 174.49, 118.59, 178.49, and 82.04. Carbon was observed, and the three sugars were identified as two cymarose and one diginose. In order to clarify the position of each of these substituents, it was confirmed that the binding to the position 3 in the order of Shimarose (cymarose), diginose, Shimarose (cymarose) through HSQC and HMBC, ketone group) is located at position 14 and the ketofuran group (ketofuran group) was confirmed that the binding to position 13. In addition, the chemical chemistry of the functional groups was determined by the correlation between the hydrogen group 8 and the methyl group 19 using the NOESY spectrum. In addition, [M + H] ⁺ ion peak was observed at m / z 793.4413 through HR-TOF-MS spectrum, and compound 1 (compound 1) was identified as a compound having a C ₄₂ H ₆₄ O ₁₄ structure having a molecular weight of 792. In nature, it was determined to be the first reported new compound. Compound 1: white and amorphous powder; HR-TOF-MS mlz 793.4413 [M + H] ⁺ (calcd for C ₄₂ H ₆₄ 0 ₁₄ + H, 793.4374); ¹ H-NMR (CD ₃ OD, 500 MHz), ¹³ C-NMR (CD ₃ OD, 125 MHz) (see FIGS. 3 and 4A).

(2) Compound 2

The NMR spectrum of Compound 2 isolated from the CA-C4-L25 active fraction showed the same behavior as Compound 1 and was determined in [M + H at m / z 793.4459 through HR-TOF-MS spectrum. ] ⁺ Ion peak was observed, Compound 2 is a compound having a C ₄₂ H ₆₄ O ₁₄ structure of molecular weight 792 was estimated to be a compound isomer and a compound isomer. Therefore, compared to compound 1, carbon 12 shifted to the low field, and the chemical stereostructure of the functional group through the correlation of hydrogen 8 and 19 and 18 methyl groups in the NOESY spectrum. By estimating (stereochemistry), Compound 2 was determined to be a compound in which the coordination of the ketofurane ring, which was bound to Compound 1 to 13, was reversed, and it was confirmed that the compound was the first reported in nature. Compound 2: white and amorphous powder; HR-TOF-MS mlz 793.4459 [M + H] ⁺ (calcd for C ₄₂ H ₆₄ O ₁₄ + H, 793.4374); ¹ H-NMR (CD ₃ OD, 500 MHz), ¹³ C-NMR (CD ₃ OD, 125 MHz). (See FIGS. 3 and 4b).

3. Whitening Activity of Column Fraction

(1) Solvent Fraction Whitening Activity of White Rice Extract

The activity model evaluated the effect of samples on melanin production in the B16 melanoma cell line stimulated with isobutylmethylxanthine (IBMX). Of the four solvent fractions of white rice extract, only MC (methylene chloride) layer (CA-MC) showed the whitening effect. The effect was 80% at sample concentration of 5 ug / ml, 24% at 2.5 ug / ml and 1.3 ug /. 8% in ml, ED ₅₀ value was 3.7 ug / ml.

TABLE 1

(2) white rice Methylene chloride (MC) solvent Fraction  Silica gel column Fraction Whitening activity

The whitening effect of CA-MC-FR-4 and CA-MC-FR-5 in 10 silica gel column chromatography fractions of the white rice methylene chloride solvent active fraction was shown. Samples CA-MC-FR-4 is a dose-which showed dependently the whitening activity, the sample at a concentration 5 ug / ml were 97%, 2.5 ug / ml 53 % In and 1.3 ug / ml 12% at, ED ₅₀ value 2.4 ug / ml. Sample CA-MC-FR-5 showed a whitening effect of 83% at a concentration of 5 ug / ml, 21% at 2.5 ug / ml and 10% at 1.3 ug / ml, with an ED ₅₀ value of 2.4 ug / ml.

TABLE 2

[Table 3]

(3) Silica Gel Column Fraction Whitening Activity of White Rice CA-MC-FR-4 Fraction

Among the 15 silica gel column fractions of white rice CA-MC-FR-4 fraction, CA-C4-L showed a whitening effect, and the ED ₅₀ value was 1.4 ug / ml.

[Table 4]

(4) Whitening Activity of ODS Column Fraction of White Rice CA-C4-L Fraction

Among the seven column fractions of the white rice CA-C4-L fraction, the whitening effect was shown in the CA-C4-L25 fraction, and the IC ₅₀ value was 1.8 ug / ml. Considering that it is higher than the IC ₅₀ value of CA-C4-L, which was a fraction before column chromatography, it is estimated that some effective ingredients were transferred to CA-C4-L26.

TABLE 5

(5) Whitening Activity of Compound 1 and Compound 2 Isolated from White Rice CA-C4-L25

Preparative HPLC was performed on the fraction CA-C4-L25 to purely separate-purify Compounds 1 and 2 as active ingredients. Compounds 1 and 2 were the major constituents in the fraction CA-C4-L25, accounting for 80% or more. Compound 1 and compound 2 dose-dependently inhibited melanin production, and the whitening ED ₅₀ values were 2.5 ug / ml and 0.9 ug / ml, respectively.

       TABLE 6

4. Fraction CA - C4 - L25 Study of mechanism of action

(1) Effect on melanin production induced by increased cellular cAMP concentration

Alpha-melanocyte stimulating hormone (MSH) binds to cellular MCRI receptors to activate adenylate cyclase, and forskolin directly activates intracellular adenylate cyclase, and IBMX (isobutylmethylxanthine) increases cAMP concentration by inhibiting phosphodiesterase, and dibutyl (db) -cAMP has a similar effect to intracellular cAMP. Melanoma B16 cells were treated with stimulants alpha-MSH (100 nM), forskolin (20 uM), IBMX (100 uM) or db-cAMP (2 mM) and sample white rice CA-C4-L25. The amount of melanin released into the medium over time was measured by absorbance at a wavelength of 405 nm. Sample fraction CA-C4-L25 dose-dependently inhibited melanin production induced by intracellular cAMP increased with each stimulant, and the ED ₅₀ value of the whitening activity was 1-2 ug / ml (FIG. 5). Reference).

(2) Effect on tyrosinase

The effects on tyrosinase were divided into protein biosynthesis and enzyme activity. Melanoma B16 cells were treated with stimulant IBMX and sample CA-C4-L25 for 48 hours. Cell extracts were electrophoresed on SDS-acrylamide gels and Western blotting followed by anti-tyrosinase antibody, anti-TRP-1 antibody or Reacted with anti-GAPDH antibody. IBMX stimulation significantly increased the amount of tyrosinase and TRP-1 protein, and sample CA-C4-L25 inhibited the biosynthesis of enzymes that were dose-dependently increased by IBMX. As expected, samples CA-C4-L25 and alpha-MSH did not affect the amount of GAPDH (see Figure 6).

Subsequently, the effects on enzyme activity against tyrosinase were investigated. Melanoma B16 cells were treated only with stimulant IBMX and incubated for 48 hours. After treatment with the sample CA-C4-L25 to the cell extract, the rate of conversion of the substrate L-Dopa to dopachrome was measured by absorbance. Sample CA-C4-L25 did not affect the enzyme activity of tyrosinase. Taken together, the sample CA-C4-L25 dose-dependently inhibited melanin production induced by increased cAMP, which is the key enzyme of the biosynthetic pathway, tyrosinase and TRP-1. It can be seen that it is due to inhibiting the biosynthesis of (see Fig. 7).

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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1 is a view schematically showing a process for separating and purifying Compounds 1 and 2 of the present invention from Cynanchum atratum extract.

2 is an HPLC chromatogram of the compounds 1 and 2 of the present invention.

3 is a ¹ H- and ¹³ C-NMR results of the compounds 1 and 2 of the present invention.

4A shows the molecular weight and chemical structure of Compound 1 of the present invention.

4B shows the molecular weight and chemical structure of Compound 2 of the present invention.

Figure 5 stimulates melanoma B16 cells with alpha-MSH (100 nM), forskolin (20 uM), IBMX (100 uM) or db-cAMP (2 mM) and 72 hours with CA-C4-L25. Shows the result of processing. The amount of melanin in the medium was determined by measuring the absorbance at 405 nm.

Figure 6 shows the results of measuring the effect of the fraction of the present invention on the biosynthesis of tyrosinase (tyrosinase). Cells were treated with IBMX and CA-C4-L25 for 48 hours. Cell lysates were electrophoresed on an SDS-acrylamide gel and transferred to a membrane. Blots were reacted with anti-tyrosinase antibodies, anti-TRP-1 antibodies or anti-GAPDH antibodies and with horseradish-conjugated secondary antibodies.

Figure 7 shows the result of measuring the effect of the fraction of the present invention on tyrosinase activity (tyrosinase). Cells were treated for 48 hours with IBMX (100 uM) alone. Cell lysates were treated with CA-C4-L25 using the enzyme source of tyrosinase. L-Dopa was used as a substrate to measure the rate at which dopachrome was formed. The tyrosinase activity of converting 1 mole of L-Dopa into dopachrome per minute was set to 1 unit.

Claims (9)

delete delete delete delete delete delete A novel pregnane compound represented by the following Chemical Formula 1 or 2, purified from Cynanchum atratum extract.

[Formula 1]

[Formula 2] (a) a cosmetically effective amount of a pregnane compound represented by Formula 1 or Formula 2 below; And (b) a cosmetically acceptable carrier.

[Formula 1]

[Formula 2] (a) a pharmaceutically effective amount of a pregnane compound represented by Formula 1 or Formula 2 below; And (b) a pharmaceutical composition for the treatment or prophylaxis of hyperpigmentation diseases comprising a pharmaceutically acceptable carrier.

[Formula 1]

[Formula 2]

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