KR102099551B1 - A composition for skin whitening comprising Dendropanax morbifera Lev. extract or a new active ingredient isolated therefrom - Google Patents

Abstract

The present invention relates to a composition for treating, alleviating, or preventing skin pigmentation disorders or symptoms, or a skin-lightening composition, comprising an extract of Dendropanax morbifera Lev. leaves and novel active ingredients isolated therefrom, as effective components. The extract of the present invention and the novel active ingredients isolated therefrom exhibit excellent effects in inhibiting melanogenesis and inhibiting the expression of melanogenesis-related proteins (MITF, Tyrosinase, TRP-1, and TRP-2) in melanoma. Accordingly, the extract of the present invention and the novel active ingredients isolated therefrom may be beneficially used as a cosmetic composition for preventing or treating various skin pigmentation disorders including hyperpigmentation. A composition having skin-lightening activity, comprising an extract of Dendropanax morbifera Lev. leaves and novel two types of single components as effective components, may be used as a pharmaceutical composition, a cosmetic composition, or a food composition capable of preventing or treating various skin pigmentation disorders including hyperpigmentation.

Description

A composition for skin whitening comprising Dendropanax morbifera Lev. extract or a new active ingredient isolated therefrom}

The present invention relates to a composition for skin whitening comprising hwangchil tree leaf extract. In addition, the present invention relates to a pharmaceutical composition, a cosmetic composition and a food composition comprising a novel whitening active substance DMW-1, DMW-2 or salts thereof separated from hwangchil tree leaf extract as an active ingredient. . The whitening composition of the present invention has excellent cell stability while having tyrosinase inhibition, melanin production inhibition, melanin production-related protein inhibition, and mechanism inhibition activity.

Melanin plays an important role in determining the color of the skin, eyes, and hair of humans and animals, and protects the skin from sunlight. Melanin is a substance produced from melanosomes present in melanocytes, tyrosinase, tyrosinase-related protein-1, and three main enzymes involved in melanin production. It is produced by tyrosinase-related protein-2.

Tyrosinase hydrolyzes tyrosine to convert it to dopa (L-DOPA) and dopa quinone, resulting in dark red-red eumelanin and tan-brown pheomelanin 2 Synthesizes melanin.

Factors such as ultraviolet light, cytokines, growth factors, and hormones affect melanin synthesis, the most important of which is α-MSH (α-melanocyte stimulating hormone). When the intracellular signal transduction is amplified by combining MC1R (melanocortin 1 receptor) and α-MSH, the downstream signaling agent PKA (protein kinase A) is activated to finally increase the expression of MITF (microphthalmia-associated transcription factor). MITF regulates melanin synthesis by inducing expression of enzymes such as tyrosinase, tyrosinase-related protein-1, and tyrosinase-related protein-2 as transcription regulatory factors specific to melanocytes.

Signaling pathways that regulate melanin production vary widely, but among them, the MAPKs signaling pathway is known to be the most important for melanin synthesis. In the case of ERK and JNK, melanin production increases as phosphorylation progresses in the initial stage, but it is known that, over time, phosphorylation is suppressed to finally inhibit melanin synthesis. In the case of p38, when phosphorylation increases, the expression of MITF increases, which plays a role in melanin synthesis. Various substances such as arbutin, kojic acid, vitamin C (ascorbic acid), and mulberry extract, which are representative melanin synthesis inhibitors, have been used as functional cosmetic ingredients. However, as the improvement of living standards and the interest in beauty and health have rapidly increased, various cosmetics using natural materials have been released and many studies have been conducted, but the situation is still insufficient.

Dendropanax morbifera Lev. Is an evergreen broad-leaved tree that grows in the southern Jeollanam-do coastal regions of Korea and in the wet areas of Jeju Island's beaches and islands. Looking at the distribution map of Korea's Hwangchil, it grows the most in the beaches such as Jeju Island, Wando, Jeonnam, Daeheksando, and Geomundo. Since ancient times, the yellow essence that comes out of a wound on a bark of a hwangchil tree was used to paint furniture, armor, weapons, and so on.

Recently, research results related to hwangchil trees revealed that hwangchil has pharmacological effects such as anti-cancer, antioxidant, anti-inflammatory, anti-obesity, anti-diabetic, antibacterial, anti-thrombotic, immune-enhancing, and arthritis-inhibiting effects. It is a trend to study in.

In hwangchil, various bioactive substances such as dendropanoxide, α-amyrin, β-amyrin, tetradecanol, and β-sistosterol ) And rutin. In addition, their anti-inflammatory, immunosuppressive, anti-obesity, anti-diabetic, antioxidant, anti-cancer pharmacological effects are known. Based on the effects revealed through various studies, Hwangchil is drawing attention as a material for functional foods and cosmetics.

Republic of Korea Patent Registration No. 10-1367306 "Whitening or moisturizing cosmetic composition comprising tetradecanol separated from hwangchil tree as an active ingredient", a single component (tetradecanol) separated from hwangchil tree extract inhibits tyrosinase activity and It is disclosed that it has a melanin production suppression and moisturizing effect.

Republic of Korea Registered Patent No. 10-1650410 "Hwangchil tree, island oak and ginseng extract composition for improving the whitening containing an extract as an active ingredient, and functional food and herbal cosmetics prepared using the same, the Hwangchil tree and It is disclosed that the complex extract has an inhibitory effect on tyrosinase activity and an inhibitory effect on melanin-producing proteins, thereby showing an excellent effect on whitening improvement.

The present inventors seek to develop a material that is safe for the human body, especially a plant-derived material, which can more effectively prevent or treat hyperpigmentation on the basis of increasing interest in beauty.

Melanin plays an important role in determining the color of the skin, eyes, and hair of humans and animals, and protects the skin from sunlight. Melanin is a substance produced from melanosomes present in melanocytes, tyrosinase, tyrosinase-related protein-1, and three main enzymes involved in melanin production. It is produced by tyrosinase-related protein-2.

Tyrosinase hydrolyzes tyrosine to convert it to dopa (L-DOPA) and dopa quinone, resulting in dark red-red eumelanin and tan-brown pheomelanin 2 Synthesizes melanin.

Factors such as ultraviolet light, cytokines, growth factors, and hormones affect melanin synthesis, the most important of which is α-MSH (α-melanocyte stimulating hormone). When the intracellular signal transduction is amplified by combining MC1R (melanocortin 1 receptor) and α-MSH, the downstream signaling agent PKA (protein kinase A) is activated to finally increase the expression of MITF (microphthalmia-associated transcription factor). MITF regulates melanin synthesis by inducing expression of enzymes such as tyrosinase, tyrosinase-related protein-1, and tyrosinase-related protein-2 as transcription regulatory factors specific to melanocytes.

Signaling pathways that regulate melanin production vary widely, but among them, the MAPKs signaling pathway is known to be the most important for melanin synthesis. In the case of ERK and JNK, melanin production increases as phosphorylation progresses in the initial stage, but it is known that, over time, phosphorylation is suppressed to finally inhibit melanin synthesis. In the case of p38, when phosphorylation increases, the expression of MITF increases, which plays a role in melanin synthesis. Various substances such as arbutin, kojic acid, vitamin C (ascorbic acid), and mulberry extract, which are representative melanin synthesis inhibitors, have been used as functional cosmetic ingredients. However, as the improvement of living standards and the interest in beauty and health have rapidly increased, various cosmetics using natural materials have been released and many studies have been conducted, but the situation is still insufficient.

Dendropanax morbifera Lev. Is an evergreen broad-leaved tree that grows in the southern Jeollanam-do coastal regions of Korea and in the wet areas of Jeju Island's beaches and islands. Looking at the distribution map of Korea's Hwangchil, it grows the most in the beaches such as Jeju Island, Wando, Jeonnam, Daeheksando, and Geomundo. Since ancient times, the yellow essence that comes out of a wound on a bark of a hwangchil tree was used to paint furniture, armor, weapons, and so on.

Recently, research results related to hwangchil trees revealed that hwangchil has pharmacological effects such as anti-cancer, antioxidant, anti-inflammatory, anti-obesity, anti-diabetic, antibacterial, anti-thrombotic, immune-enhancing, and arthritis-inhibiting effects. It is a trend to study in.

In hwangchil, various bioactive substances such as dendropanoxide, α-amyrin, β-amyrin, tetradecanol, and β-sistosterol ) And rutin. In addition, their anti-inflammatory, immunosuppressive, anti-obesity, anti-diabetic, antioxidant, anti-cancer pharmacological effects are known. Based on the effects revealed through various studies, Hwangchil is drawing attention as a material for functional foods and cosmetics.

Republic of Korea Patent Registration No. 10-1367306 "Whitening or moisturizing cosmetic composition comprising tetradecanol separated from hwangchil tree as an active ingredient", a single component (tetradecanol) separated from hwangchil tree extract inhibits tyrosinase activity and It is disclosed that it has a melanin production suppression and moisturizing effect.

Republic of Korea Registered Patent No. 10-1650410 "Hwangchil tree, island oak and ginseng extract composition for improving the whitening containing an extract as an active ingredient, and functional food and herbal cosmetics prepared using the same, the Hwangchil tree and It is disclosed that the complex extract has an inhibitory effect on tyrosinase activity and an inhibitory effect on melanin-producing proteins, thereby showing an excellent effect on whitening improvement.

The present inventors seek to develop a material that is safe for the human body, especially a plant-derived material, which can more effectively prevent or treat hyperpigmentation on the basis of increasing interest in beauty.

One aspect of the present invention relates to a composition for treating, improving or preventing skin pigmentation disease or symptoms comprising an extract of Dendropanax morbifera Lev. Leaf.

The present inventors, as a result of diligent research efforts to develop a plant-derived composition for skin whitening that inhibits melanin production, include a new whitening active substance DMW-1 or DMW-2 separated from a hwangchil leaf extract or hwangchil leaf extract The skin whitening or skin hyperpigmentation inhibiting composition was developed.

The present inventors have found that five single components isolated from hwangchil tree leaf extract and extract inhibit the accumulation of activated melanin and have excellent whitening effect and are very effective in preventing or treating. Among them, the new substances DMW-1 and DMW-2 showed the best efficacy.

In addition, when treated with a mixed composition (especially, when mixed at a ratio of 1: 1) of the Hwangchil tree leaf ( Dendropanax morbifera Lev. Leaf) and the brown flower ( Puerariae Flos ), each extract was intracellularly treated than when treated separately. It was found that the inhibitory effect of tyrosinase activity was more excellent.

The whitening active substances isolated from the extract of Hwangchil tree ( Dendropanax morbifera Lev. Leaf) are as follows.

DMW-1: methyl (10E) -9,16-dihydroxyoctadeca-10,17-dien-12,14-diynoate; Methyl (10E) -9,16-dihydroxyoctadeca-10,17-diene-12,14-diinoate,

DMW-2: (10E)-(-)-10,17-Octadecadiene-12,14-diyne-1,9,16-triol; (10E)-(-)-10,17-octadecadiene-12,14-diyne-1,9,16-triol.

As used herein, the term "skin pigmentation disease" refers to a disease or symptom related to melanin hyperplasia, wherein the skin pigmentation disease or symptom is tinge, freckles, liver spots, pigmentation changes due to skin aging, spots or brown spots, low pigmentation Deposition and hyperpigmentation, but is not limited thereto.

According to one embodiment of the invention, the hyperpigmentation is post-inflammatory hyperpigmentation, or hyperpigmentation after drug use due to wounds or dermatitis.

The term "comprising as an active ingredient" in the present specification means to include an amount sufficient to achieve the efficacy or activity of the hwangchil tree leaf extract. Quantitative upper limit of the hwangchil tree leaf extract contained in the composition of the present invention can be carried out by selecting within a suitable range by those skilled in the art. According to one embodiment of the present invention, the composition of the present invention may be implemented as a pharmaceutical composition, a cosmetic composition or a food composition comprising a hwangchil tree leaf extract as an active ingredient.

Hereinafter, the present invention will be described in more detail.

The hwangchil tree leaf extract of the present invention is dried and shredded hwangchil tree leaf ( Dendropanax morbifera Lev. Leaf), and then added a polar solvent (for example, distilled water, methanol, ethanol, propanol, butanol, etc.) as an extraction solvent, 80 It can be extracted by heating to ℃ -120 ℃. According to one embodiment of the invention, it can be extracted by heating to 80 ℃ -120 ℃ for 2-8 hours.

The polar solvent used in the extraction method of the present invention includes (i) water, (ii) alcohol (preferably methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, normal-butanol), (iii) Acetic acid, (iv) dimethyl-formamide (DMFO) and (v) dimethyl sulfoxide (DMSO). According to an embodiment of the present invention, the polar solvent is one or more solvents selected from the group consisting of water and lower alcohol having 1 to 3 carbon atoms. According to another embodiment of the invention, the polar solvent is water, methanol or ethanol.

According to one embodiment of the present invention, a polar solvent may be added to the hwangchil tree leaves to extract at 90 ° C-110 ° C for 3-5 hours.

According to a specific embodiment of the present invention, a polar solvent may be added to hwangchil leaves to extract at 95 ° C-105 ° C for 3-5 hours.

According to a specific embodiment of the present invention, it can be extracted for 4 hours at 95 ℃ -105 ℃ by adding a polar solvent to the hwangchil tree leaves.

In the present invention, when extracting hwangchil tree leaves, hot water extraction, reflux extraction, room temperature extraction, or ultrasonic extraction may be used. According to one embodiment of the present invention, a hot water extraction method or a reflux extraction method may be used.

According to a specific embodiment of the present invention, the hwangchil tree leaves were extracted with distilled water, filtered, concentrated under reduced pressure, and dried.

In the present invention, extract of hwangchil tree leaves with distilled water, it was confirmed that the extract inhibits intracellular tyrosinase activity and melanin accumulation induced by α-MSH in B16-F10 cells, which are melanoma cell lines. In addition, hwangchil tree leaf extract inhibited the expression of melanin-related proteins, MITF, tyrosinase, TRP-1, and TRP-2. Hwangchil tree leaf extract inhibited the accumulation of melanin by inhibiting phosphorylation of MAPKs p38 among various mechanisms involved in melanin production.

Meanwhile, the composition of the present invention may further include an extract of Puerariae Flos .

The gallium extract of the present invention is dried and crushed brown gallium ( Puerariae Flos ), and then added with a polar solvent (for example, distilled water, methanol, ethanol, etc.) as an extraction solvent, refluxed at 60 ℃ -100 ℃ for 2-8 hours. Can be obtained. Polar solvents used in the production of gallium extract are as described above.

According to one embodiment of the present invention, a polar solvent is added to browning to extract at 70 ° C-90 ° C for 3-5 hours.

According to a specific embodiment of the present invention, water or methanol may be added to browning to extract at 75 ° C-85 ° C for 3-5 hours.

According to a specific embodiment of the present invention, water or methanol may be added to browning to extract at 75 ° C-85 ° C for 4 hours.

In the present invention, when extracting browning, hot water extraction, reflux extraction, room temperature extraction, or ultrasonic extraction may be used. According to one embodiment of the present invention, a hot water extraction method or a reflux extraction method may be used.

The composition for treating, improving or preventing skin pigmentation disease or symptom of the present invention may include an extract obtained by mixing a water extract of hwangchil tree leaf and a water extract of galaxia in a weight ratio of 1: 1.

Alternatively, the composition for the treatment, improvement or prevention of skin pigmentation disease or symptom of the present invention may include an extract obtained by mixing a water extract of Hwangchil tree and a water extract of galaxia in a concentration ratio of 1: 1 (w / v).

According to an embodiment of the present invention, when treated with 0.3 mg / ml (0.03%) of Hwangchil tree leaf water extract and brown water extract, respectively, it was found that the inhibitory effect on intracellular tyrosinase activity is the best.

In addition, the extract of the present invention includes not only the extract by the above-described extraction solvent, but also an extract that has undergone a conventional purification process. Obtained through various purification methods additionally performed, such as separation using an ultrafiltration membrane having a constant molecular weight cut-off value, and separation by various chromatography (made for separation according to size, charge, hydrophobicity or affinity) Fractions are also included in the extract of the present invention.

The extract of the present invention may be prepared in powder form by additional processes such as distillation under reduced pressure and freeze drying or spray drying.

Another aspect of the present invention relates to a composition for skin whitening comprising an extract of hwangchil tree leaf ( Dendropanax morbifera Lev. Leaf).

Another aspect of the present invention relates to a composition for external application for skin comprising an extract of hwangchil tree leaf ( Dendropanax morbifera Lev. Leaf).

Another aspect of the present invention relates to a composition for skin whitening comprising a compound represented by the following Chemical Formula 1 and Chemical Formula 2, and one or more compounds selected from the group consisting of salts thereof:

[Formula 1]

; 및

; And

[Formula 2]

.

.

The present inventors isolated five single components from the extract of Hwangchil tree ( Dendropanax morbifera Lev. Leaf). The linoleic acid, trilinolein, and methyl chlorogenate were separated from the new substances DPW1 and DPW2, and their intracellular tyrosinase inhibition and melanin production inhibitory effects were confirmed. Came to the invention.

Among the components, the compound names of DMW-1 and DMW-2 are as follows:

DMW-1: methyl (10E) -9,16-dihydroxyoctadeca-10,17-dien-12,14-diynoate; Methyl (10E) -9,16-dihydroxyoctadeca-10,17-diene-12,14-diinoate,

DMW-2: (10E)-(-)-10,17-Octadecadiene-12,14-diyne-1,9,16-triol; (10E)-(-)-10,17-octadecadiene-12,14-diyne-1,9,16-triol.

The composition for skin whitening of the present invention relates to a composition having a whitening activity containing two single components of DMW-1 and / or DMW-2 as an active ingredient. DMW-1 and DMW-2, new substances isolated from hwangchil tree leaf extract, inhibit intracellular tyrosinase activity in melanoma, inhibit melanin production, and melanin-related proteins MITF, tyrosinase, and tyrosinase-associated It suppresses the expression of protein-1 (TRP-1), tyrosinase-associated protein-2 (TRP-2), and inhibits the phosphorylation of p38 of MAPKs, one of the mechanisms of melanin production. Therefore, it can be usefully used as a composition that can prevent or treat various pigmentation symptoms or diseases, including hyperpigmentation.

Methyl (10E) -9,16-dihydroxyoctadeca-10,17-diene-12,14-diinoate and (10E)-(-)-10,17-octa included in the composition of the present invention Decadiene-12,14-diyne-1,9,16-triol is a compound isolated from the natural plant material, Dendropanax morbifera Lev. Leaf. The upper limit of the amount of the compound or salts included in the composition of the present invention can be carried out by a person skilled in the art within a suitable range. According to one embodiment of the invention, the composition of the invention is methyl (10E) -9,16-dihydroxyoctadeca-10,17-diene-12,14-diinoate, (10E)-(- ) -10,17-octadecadiene-12,14-diyne-1,9,16-triol, or salts thereof as an active ingredient.

The compound used as an active ingredient in the present invention may be used by itself or in the form of salts, preferably in the form of pharmaceutically acceptable salts. The salt is preferably an acid addition salt formed by free acid. As the free acid, an organic acid and an inorganic acid can be used. The organic acid is citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, glutanoic acid and aspartic acid, etc. It includes, but is not limited to. In addition, the inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid.

Methyl (10E) -9,16-dihydroxyoctadeca-10,17-diene-12,14-diinoate and (10E)-(-)-10,17-octa included in the composition of the present invention Decadiene-12,14-diyne-1,9,16-triol is a compound isolated from the natural plant material, Dendropanax morbifera Lev. Leaf. Hwangchil tree leaves have been used for edible and folk medicine for a long time. Since it has come, it can be expected that the compound of the present invention separated therefrom will also have no problems such as toxicity and side effects. Thus, methyl (10E) -9,16-dihydroxyoctadeca-10,17-diene-12,14-diinoate and (10E)-(-)-10,17-octadecadiene-12, 14-Diyne-1,9,16-triol, or salts thereof, can be used as pharmaceutical, food, and cosmetic compositions.

According to one embodiment of the present invention, the composition of the present invention may be a pharmaceutical composition.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is commonly used in the preparation, 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, but is not limited thereto. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, and for parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, and the like.

Suitable dosages of the pharmaceutical compositions of the present invention vary by factors such as formulation method, mode of administration, patient's age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion, and response sensitivity, Usually, a skilled physician can easily determine and prescribe a dose effective for the desired treatment or prevention. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-10000 mg / kg.

The pharmaceutical composition of the present invention is prepared in a unit dose form by formulating using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily carried out by those skilled in the art to which the present invention pertains. Or it can be manufactured by incorporating into a multi-dose container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of ex, powder, granule, tablet, or capsule, and may further include a dispersant or stabilizer.

According to another embodiment of the present invention, the composition of the present invention may be a cosmetic composition.

The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleaning , May be formulated as an oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, skin lotion, skin toner, lotion, moisture lotion, nutrition cream, massage cream, moisture cream, hand cream, foundation, essence, nutrition essence, shampoo, conditioner, soap, cleansing foam, cleansing oil, cleansing lotion, Cleansing tissues, body creams, body lotions, body cleansers, packs, sprays and powders can be prepared in any one formulation selected from the group consisting of.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, trakant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide, etc. may be used as a carrier component. You can.

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

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

When the formulation of the present invention is a suspension, liquid diluents such as water, ethanol or propylene glycol as carrier components, ethoxylated isostearyl alcohol, suspensions such as polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, microcrystals Sex cellulose, aluminum metahydroxide, bentonite, agar or trakant, etc. can be used.

When the formulation of the present invention is surfactant-containing cleansing, as the carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivatives, 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 can be used.

The ingredients included in the cosmetic composition of the present invention include ingredients commonly used in cosmetic compositions, in addition to the active ingredients and carrier ingredients, and include conventional auxiliary agents such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances. It can contain. In the cosmetic composition of the present invention, the amount of the compound represented by Formula 1 as an active ingredient or salts thereof is not particularly limited, and is preferably included in an amount sufficient to achieve the wrinkle improvement and moisturizing effect.

According to another embodiment of the present invention, the composition of the present invention may be a food composition.

When the composition of the present invention is a food composition, it may be prepared in the form of powder, granule, tablet, capsule or beverage. For example, there are various foods such as candy, beverages, gum, tea, vitamin complexes, and health supplements.

Food composition of the present invention, as an active ingredient hwangchil tree leaves ( Dendropanax morbifera Lev. Leaf) extract, methyl (10E) -9,16-dihydroxyoctadeca-10,17-diene-12,14-diinoate , (10E)-(-)-10,17-octadecadiene-12,14-diyne-1,9,16-triol, or salts thereof, as well as commonly added in food manufacturing Ingredients may include, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents. Examples of the above-mentioned carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose, sucrose, oligosaccharides, etc .; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a flavoring agent, natural flavoring agents (tauumatin, stevia extract (eg, rebaudioside A, glycyrrhizine, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is prepared as a drink agent, in addition to the extract or the compound of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, worm extract, jujube extract, licorice extract, etc. You can.

Another aspect of the present invention, the treatment of the pigmentation diseases or symptoms of skin, including one or more compounds selected from the group consisting of compounds represented by Formula 1 and Formula 2, and salts (salts) thereof The composition relates to:

[Formula 1]

; 및

; And

[Formula 2]

.

.

The composition for treating, improving or preventing skin pigmentation disease or symptom of the present invention uses the same active ingredient as the composition for skin whitening of the present invention described above, and the common content between the two is to avoid excessive complexity of the present specification. , The description is omitted.

Another aspect of the present invention relates to a composition for external application for skin comprising at least one compound selected from the group consisting of the compounds represented by Chemical Formulas 1 and 2 and salts thereof.

Another aspect of the present invention includes the step of administering to the subject a composition comprising at least one compound selected from the group consisting of compounds represented by Formula 1 and Formula 2, and salts thereof. It relates to a method for treating, suppressing or improving skin pigmentation.

The term "administration" as used herein means providing a given substance to a subject in any suitable way. The route of administration of the composition for treating, inhibiting or improving skin pigmentation of the present invention can be administered orally or parenterally through any general route as long as it can reach the target tissue. In addition, the composition of the present invention may be administered using any device capable of delivering an active ingredient to target cells.

The term "subject" herein is not particularly limited, for example, human, monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit, or Guinea pigs, preferably mammals, more preferably humans.

Another aspect of the present invention relates to the skin whitening, skin pigmentation treatment, suppression or improvement use of one or more compounds selected from the group consisting of the compounds represented by Formula 1 and Formula 2, and salts thereof. will be.

Another aspect of the present invention relates to a compound represented by the following Chemical Formula 1 or salts thereof:

[Formula 1]

Another aspect of the invention, methyl (10E) -9,16-dihydroxyoctadeca-10,17-diene-12,14-diinoate or (10E)-(-) comprising the following steps: -10,17-octadecadiene-12,14-diyne-1,9,16-triol relates to a method of separation of the compound:

(a) extracting hwangchil tree leaves ( dendropanax morbifera lev . leaf) with a polar solvent;

(b) purifying only the extract soluble in the ethyl acetate solvent from the hwangchil tree leaf extract of step (a) to obtain an ethyl acetate extract fraction; And

(c) Ethyl acetate extract fractions were purified to give methyl (10E) -9,16-dihydroxyoctadeca-10,17-diene-12,14-diinoate or (10E)-(-)-10, Obtaining 17-octadecadiene-12,14-diyne-1,9,16-triol.

The whitening active substances isolated from the extract of Hwangchil tree ( Dendropanax morbifera Lev. Leaf) are as follows.

DMW-1: methyl (10E) -9,16-dihydroxyoctadeca-10,17-dien-12,14-diynoate; Methyl (10E) -9,16-dihydroxyoctadeca-10,17-diene-12,14-diinoate,

DMW-2: (10E)-(-)-10,17-Octadecadiene-12,14-diyne-1,9,16-triol; (10E)-(-)-10,17-octadecadiene-12,14-diyne-1,9,16-triol.

Hereinafter, the method for separating the compound will be described in detail.

Step (a): Hwangchil tree leaves ( Dendropanax morbifera Lev. leaf)

First, a polar solvent is added to the hwangchil tree leaves ( dendropanax morbifera lev . Leaf) to extract.

The method for obtaining the hwangchil tree leaf extract is as described above.

According to one embodiment of the present invention, the polar solvent is water or a low alcohol having 1 to 3 carbon atoms.

According to a specific embodiment of the present invention, water or methanol may be added to the hwangchil tree leaves and heated to 80 ° C-120 ° C to obtain an extract.

Step (b): Preparation of ethyl acetate extracted fraction

Subsequently, by extracting only the extract soluble in the ethyl acetate solvent from the hwangchil tree leaf extract of step (a), an ethyl acetate extract fraction is obtained.

According to one embodiment of the present invention, after adding water of about 0.001 to 100 times, preferably 1 to 70 times, more preferably 50 times the volume (v / w%) of the weight of hwangchil tree leaf extract, hexane, chloroform , By performing a conventional fractionation process using a non-polar solvent such as ethyl acetate, a non-polar solvent-soluble extract fraction soluble in the non-polar solvent can be obtained.

The non-polar solvent used in the separation method of the present invention is hexane, chloroform, ethyl acetate, acetone, acetonitrile, methyl acetate, fluoroalkane, pentane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane , Diisobutylene, 1-pentene, 1-chlorobutane, 1-chloropentane, o-xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene, benzene, diethyl ether , Diethyl sulfide, dichloromethane, 1,2-dichloroethane, anneal, diethylamine, ether, carbon tetrachloride and THF. According to one embodiment of the invention, the non-polar solvent is hexane, chloroform, ethyl acetate or dichloromethane. According to a specific embodiment of the present invention, in step (b), as a non-polar organic solvent, (i) hexane, chloroform, and ethyl acetate are sequentially used to obtain a distribution fraction; Or (ii) dichloromethane and ethyl acetate are used sequentially to obtain a partitioned fraction.

On the other hand, after obtaining a fraction using a non-polar solvent, a polar solvent-soluble extract fraction soluble in a polar solvent (for example, butanol, water, etc.) can be obtained from the remaining hwangchil tree leaf extract.

The polar solvent used in the separation method of the present invention is (i) water, (ii) alcohol (preferably, methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, normal-butanol, 1-pentanol , 2-butoxyethanol or ethylene glycol), (iii) acetic acid, (iv) DMFO (dimethyl-formamide) and (v) DMSO (dimethyl sulfoxide). According to one embodiment of the invention, the polar solvent is water or alcohol. According to a specific embodiment of the present invention, after adding butanol to the hwangchil tree leaf extract, a fraction of the butanol layer and the aqueous layer was obtained.

According to one embodiment of the invention, step (b) may be carried out as follows:

(b-1) separating and removing extract fractions containing only extracts soluble in at least one non-polar solvent selected from the group consisting of dichloromethane, hexane, and chloroform from the hwangchil tree leaf extract of step (a); And

(b-2) Separating the extracted fraction of step (b-1) and remaining hwangchil tree leaf extract

The step of obtaining an ethyl acetate extract fraction containing only the extract soluble in the ethyl acetate solvent.

Step (c): Methyl (10E) -9,16-dihydroxyoctadeca-10,17-diene-12,14-diinoate or (10E)-(-)-10,17-octadecadiene -12,14-diyne-1,9,16-triol tablets

Then, the ethyl acetate extract fraction obtained in step (b) is purified to obtain each compound.

According to an embodiment of the present invention, in the purification step (c), various known purification methods can be used, for example, column chromatography and thin layer chromatography.

According to an embodiment of the present invention, after the ethyl acetate extract fraction of step (b) is loaded onto a column, column chromatography purification may be performed using a solvent gradient of a non-polar solvent-polar solvent.

According to one embodiment of the present invention, a purified ethyl acetate fraction can be obtained using a solvent gradient of chloroform-methanol.

The purified ethyl acetate fraction can be separated from the compound DMW-1 by repeating column chromatography with a mobile phase in a polar solvent. According to one embodiment of the present invention, the purified ethyl acetate fraction was subjected to YMC RP-18 column chromatography repeatedly to separate compound DMW-1.

According to an embodiment of the present invention, methanol-water may be used as a polar organic solvent.

On the other hand, the purified ethyl acetate fraction can be separated from the compound DMW-2 by repeating column chromatography with a non-polar solvent as a mobile phase. According to one embodiment of the present invention, the purified ethyl acetate fraction was subjected to silica gel and YMC RP-18 column chromatography repeatedly to separate compound DMW-2.

According to one embodiment of the present invention, hexane-acetone may be used as a non-polar organic solvent.

The polar solvent or non-polar solvent usable in step (c) is as described above.

The features and advantages of the present invention are summarized as follows:

The present invention relates to a composition for treating, improving or preventing skin pigmentation diseases or symptoms containing a hwangchil tree leaf extract and a new active ingredient separated therefrom as an active ingredient, or a composition for skin whitening.

The extract of the present invention and the new active ingredient isolated therefrom had excellent melanin production inhibition and melanin production-related proteins (MITF, Tyrosinase, TRP-1, TRP-2) in melanoma production. Therefore, it can be usefully used as a cosmetic composition for preventing or treating various pigmentation diseases including hyperpigmentation.

The composition having a whitening activity containing hwangchil tree leaf extract and two single components as an active ingredient may be used as a pharmaceutical composition, cosmetic composition, or food composition capable of preventing or treating various pigmentation diseases including hyperpigmentation.

Figure 1 shows the effect on the survival rate according to the presence or absence of α-MSH in B16-F10 cells by concentration of hwangchil tree water extract (Dm).
Figure 2 shows the degree of inhibiting the activity of intracellular tyrosinase (intracellular tyrosinase) produced by α-MSH in B16-F10 cells by concentration of water extract (Dm) hwangchil tree.
3a-3b shows the degree of melanin production inhibition by α-MSH in B16-F10 cells according to the concentration of water extract (Dm) of hwangchil tree leaves. (3a) melanin content; (3b) Photograph of cell pellet change.
4A-4B show the degree of inhibition of a protein related to the production of melanin activated by α-MSH in H16 leaf water extract (Dm) in B16-F10 cells. (4a) proteins of MITF, tyrosinase, TRP-1, TRP-2; (4b) MITF, tyrosinase, TRP-1, TRP-2 quantification graph.
5A-5F show inhibition of phosphorylation of the MAPKs protein in which Hwangchil tree leaf water extract (Dm) is involved in the whitening-related mechanism induced by α-MSH. MAPKs protein phosphorylation is inhibited by hwangchil tree leaf extract. (5a) proteins of MAPKs (ERK, JNK, p38); (5b) MAPKs quantification graph; (5c) Confirmation of changes in tyrosinase activity in cells through treatment with specific inhibitors; (5d) Confirmation of melanin production change through specific inhibitor treatment; (5e) Confirmation of changes in intracellular tyrosinase activity through treatment with hwangchil leaf extract (Dm) and p38 inhibitors; (5f) Confirmation of melanin production change through treatment with hwangchil leaf extract (Dm) and p38 inhibitor.
FIG. 6 shows five single components, Linolenic acid, Trilinolein, methyl chlorogenate, and new substances DMW1 and DMW2 separated from Hwangchil tree leaf water extract (Dm). The degree of inhibition of melanin production.
7A-7D are graphs showing inhibition of intracellular tyrosinase activity and inhibition of melanin production by DMW1 and DMW2, the most effective new substances among the five single components isolated from the water extract (Dm). (7a) intracellular tyrosinase activity (7b) melanin amount; (7c) Photograph of cell pellet change; (7d) Structure of Hwangchil whitening active ingredients DMW-1 and DMW-2.
FIG. 8 shows the degree of inhibition of tyrosinase activity in cells when the Hwangchil leaf extract (Dm) and the gallium extract (Pf) were treated alone or mixed (1: 1, 3: 1).

Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Throughout this specification, "%" used to indicate the concentration of a specific substance, unless otherwise specified, solids / solids (weight / weight)%, solids / liquids (weight / volume)%, and The liquid / liquid is (volume / volume)%.

Example 1: Preparation of extract

1-1. Hwangchil tree leaf water extract (Dm)

4 L of extraction solvent (distilled water) was added to 200 g of hwangchil tree leaves (Jangheung, Jeollanam-do, Korea) and refluxed at 100 ° C for 4 hours to obtain each extract. After filtering the obtained extract with filter paper, the filtrate was concentrated under reduced pressure with a rotary vacuum concentrator.

Then, each extract obtained by lyophilization was dissolved in DPBS at a concentration of 10 mg / mL, followed by syringe filtering (0.22 μm). This was used as an experimental sample (Hwangchil leaf water extract: Dm).

1-2. Galhwa Water Extract (Pf)

1L extraction solvent (distilled water) was added to 100 g of Galhwa (Yeongju, Gyeongsangbuk-do, Korea), refluxed at 80 ° C for 4 hours, and extracted twice to obtain an extract. After filtering the obtained extract with filter paper, the filtrate was concentrated under reduced pressure with a rotary vacuum concentrator. Then, each extract obtained by freeze-drying was dissolved in DPBS at a concentration of 10 mg / mL, followed by syringe filtering (0.22 μm). This was used as an experimental sample (gallium extract: Pf).

1-3. Hwangchil tree leaf and brown mixed extract

Hwangchil tree leaf water extract (Dm) and brown water extract (Pf) were mixed in an amount of 0.03% or 0.01% by weight, respectively, based on the total composition, and the mixed weight ratio of each extract was set to 1: 1 or 1: 3, respectively.

Example 2: Isolation of compounds

3.0 kg of dried and shredded hwangchil leaves ( Dendropanax Morbifera Leveille leaf ) were extracted with reflux three times for 3 hours with 10 L of 100% methanol. Then, the extract obtained by filtration of the extract was concentrated under reduced pressure to obtain 470 g of methanol extract.

2 L of distilled water and 2 L of dichloromethane were added to 470 g of the obtained methanol extract, and shaken to fractionate into a dichloromethane layer (A) and an aqueous layer (B). Thereafter, the dichloromethane layer was concentrated under reduced pressure to obtain 159 g of a dichloromethane fraction.

After adding 2 L of ethyl acetate to 2 L of the fractionated aqueous layer (B), shaking and dividing it into an ethyl acetate layer and a water layer, the ethyl acetate layer was concentrated under reduced pressure to obtain 21 g of ethyl acetate fraction.

After adding 2 L of butanol to 2 L of the fractionated aqueous layer (B), shaking and dividing it into a butanol layer and a water layer, the butanol layer and the water layer were respectively concentrated under reduced pressure to obtain 56 g of butanol fraction and 145 g of soluble fraction.

After loading the obtained ethyl acetate fraction on a silica gel column, a solvent gradient using chloroform and methanol as the mobile phase (chloroform-methanol; 1: 0, 60: 1, 30: 1, 15: 1, 10: 1, 7: Elution with 1, 5: 1, 3: 1, 1: 1, 0: 1) yielded 10 ethyl acetate fractions (EA1 to EA10).

After loading 900 mg of the obtained EA3 fraction on a silica gel column, 8 fractions (EA3A to EA3H) were eluted by gradient elution with a solvent gradient (hexane-ethyl acetate; 3: 1 to 1: 6) using hexane and ethyl acetate as mobile phases. Got.

YMC RP-18 column chromatography of 70 mg of the obtained EA3C fraction with MeOH-water (3: 1) was repeated to obtain 5.3 mg of compound DMW-1.

238 mg of the obtained EA3D fraction was repeatedly subjected to silica gel and YMC RP-18 column chromatography with n-hexane-acetone (1: 1) to obtain 6.2 mg of compound DMW-2.

Example 3: Structural analysis of compounds DMW-1 and DMW-2

HR-MS spectrum of the compound DMW-1 obtained in Example 2 (Agilent 6530 accurate mass QTOF LC / MS) was measured ([M + NH4] + m / z 336.2167, calcd for 336.2175), C ₁₉ H ₂₆ The molecular formula of O ₄ was estimated. Through the 1H-NMR, 13C-NMR, HMBC, HSQC, and COSY spectra, the compound DMW-1 was identified as a novel substance that was first isolated from nature. DMW-1 was identified as a new polyacetylene compound, methyl (10E) -9,16-dihydroxyoctadeca-10,17-dien-12,14-diynoate.

The HR-MS spectrum of the compound DMW-2 was measured ([M + HCOO] -m / z 335.1872, calcd for 335.1858) to estimate the molecular formula of C ₁₈ H ₂₆ O ₃ . Fruticotriol, (10E)-(-)-10,17-octadecadiene-12,14-diyne-1,9,16-triol via 1H-NMR, 13C-NMR, HMBC, HSQC and COSY spectra It was found to be.

Table 1 below shows the physical properties of the compounds DMW-1 and DMW-2.

- - Compound 1 - Compound 2 location δ _C δ _H ( J in Hz) δ _C δ _H ( J in Hz) One 176.0 - 63.0 3.56, t (6.7) 2 34.8 2.34, t (7.5) 33.7 1.59 *, m 3 26.0 1.62, m 26.9 1.37 *, m 4 30.4 1.35 *, m 30.7 1.37 *, m 5 30.3 1.35 *, m 30.6 1.37 *, m 6 30.1 1.35 *, m 30.5 1.37 *, m 7 26.3 1.43 *, m 26.4 1.54 *, m 8 37.8 1.51, dt (11.7, 3.6) 37.8 1.53 *, m 9 72.4 4.13, dtd (7.2, 5.7, 1.6) 72.5 4.14, m 10 151.9 6.34, dd (15.9, 5.7) 151.9 6.34, dd (15.9, 5.6) 11 108.4 5.79, ddd (15.9, 1.6, 0.9) 108.4 5.79, ddd (15.9, 1.6, 0.8) 12 78.0 - 78.0 - 13 74.0 - 74.0 - 14 70.6 - 70.6 - 15 82.2 - 82.2 - 16 64.0 4.96, dd (5.4, 1.1) 64.0 4.93, dd (5.5, 1.1) 17 138.1 5.94, ddd (17.1, 10.2, 5.4) 138.1 5.95, ddd (17.0, 10.2, 5.5) 18 116.6 5.42, d (17.1) 5.22, d (10.2) 116.6 5.43, d (17.1)
5.22, d (10.2) OCH ₃ 52.0 3.67, s - -

* Signals overlap.

Experimental Example 1: Cell culture

Mouse melanoma cells B16-F10 were purchased from a Korean cell line bank and passaged in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS, 100 U / mL penicillin and 100 μg / mL streptomycin. Cells were cultured in a 37 ° C., 5% CO ₂ incubator.

Experimental Example 2: Cytotoxicity test

Cytotoxicity test was conducted by MTS assay, and B16-F10 cells (1 X 10 ⁴ cells / well) were cultured overnight, and samples were treated for 72 hours depending on the presence or absence of α-MSH by concentration. 20 μL of MTS reagent was added to the culture solution, and absorbance was measured at 490 nm after 2 hours using a microplate spectrophotometer (BioTek Instruments, Inc., VT, USA).

As a result of the cytotoxicity test, it was shown that Hwangchil leaf extract (Dm) treated by concentration did not inhibit cell growth with or without α-MSH. That is, it was confirmed that there is no cytotoxicity (see Figure 1).

Experimental Example 3: Intracellular tyrosinase activity inhibition test

Measurement of tyrosinase in cells was confirmed by treating tyrosinase in cell lysate with L-DOPA (2 mg / mL). B16-F10 cells were dispensed in a 6-well plate so that the number of cells was 1 X 10 ⁵ cells / well and cultured for 24 hours.

Samples were treated with the culture medium for 2 hours before concentration, and tyrosinase activation was induced with α-MSH (200 nM) for 72 hours. After incubation, the cells were washed once with DPBS, and a buffer added with phosphatase inhibitor cocktail to lysis buffer (25mM Tris pH 7.5 with H3PO4,2mM CDTA, 1% TritonX-100,2mM DTT, 10% glycerol) was used. To prepare a cell lysate (cell lysate). After quantifying the protein with the Bradford reagent, 50 μg of protein and L-DOPA were mixed and incubated for 1 hour in an incubator at 37 ° C. The DOPA chrome produced at 490 nm was then measured using a microplate spectrophotometer.

The whitening effect was confirmed by the ability to inhibit tyrosinase activity in the cell. As a result, the intracellular tyrosinase of the cells treated with α-MSH increased compared to the control group not treated with α-MSH. The hwangchil leaf extract (Dm) sample inhibited tyrosinase activity by α-MSH in a dose-dependent manner (see FIG. 2).

Experimental Example 4: Melanin production inhibition experiment

B16-F10 cells were dispensed in a 6-well plate so that the number of cells was 1 X 10 ⁵ cells / well and cultured for 24 hours. Samples were treated with the culture medium for 2 hours before concentration, and melanin production was induced with α-MSH (200 nM) for 72 hours. Thereafter, the cells were washed once with DPBS, treated with trypsin-EDTA, and then collected and centrifuged. The pellet was treated with 1N NaOH containing 10% DMSO and reacted at 60 ° C. for 1 hour. Thereafter, the amount of melanin produced at 405 nm was measured using a microplate spectrophotometer.

As a result of confirming the inhibitory effect on melanin production, the melanin content of the cells treated with α-MSH increased when compared to the control group not treated with α-MSH. The hwangchil leaf extract (Dm) sample inhibited melanin production by α-MSH in a dose-dependent manner (see FIGS. 3A-3B). Figure 3b is a photograph showing the color change of the pellet.

Experimental Example 5: Melanin-related protein, MITF, Tyrosinse, TRP-1, TRP-2 expression inhibition efficacy evaluation (Western-blotting)

Measurement of protein expression patterns related to intracellular melanin production was performed by applying Western blot analysis.

In order to investigate the melanin production inhibitory effect of Hwangchilmul extract, MITF (microphthalmia-associated transcription factor), tyrosinase, TRP-1 (tyrosinase-related protein-1), TRP-2 (tyrosinase-related protein-2) ) Was measured for the amount of protein.

B16-F10 cells were dispensed in a 6-well plate so that the number of cells was 1.5 X 10 ⁵ cells / well and cultured for 24 hours. After the sample was treated with the culture solution (Hwangchilmul extract: 30, 100, 300 μg / mL) and the control drug (arbutin: 2 mM) for 2 hours, α-MSH (200 nM) was treated and cultured for 24 hours. After incubation, the cells were washed once with DPBS, and a buffer added with a phosphatase inhibitor cocktail to a lysis buffer (25 mM Tris pH 7.5 with H3PO4, 2 mM CDTA, 1% TritonX-100, 2 mM DTT, 10% glycerol) was used. Cell lysates were prepared.

The supernatant obtained by centrifuging the cell lysate at 13,000 rpm at 4 ° C. for 10 minutes was used as a total protein extract. Proteins were quantified by Bradford assay (Bio-Rad Laboratories, CA, USA). 20 μg of the quantified protein was boiled at 100 ° C. for 10 minutes, and then electrophoresed at 100 V on 10% SDS-PAGE.

Protein was attached to PVDF, blocked with 5% skim milk, and reacted with primary antibodies of MITF, Tyrosinse, TRP-1, and TRP-2, respectively. It was then treated with HRP-conjugated anti-mouse antibody as a secondary antibody. ECL (enhancedchemiluminescence, Advansta Inc., CA, USA) was used as a substrate to identify the target protein. The membrane was developed using a C300 chemiluminescence imager (Azure Biosystems, Inc., CA, USA).

Figure 4a-4b shows the results confirming the inhibitory activity of the MITF, Tyrosinse, TRP-1, TRP-2 protein induced by α-MSH of hwangchilmul extract (Dm). It was confirmed that the expression of MITF, Tyrosinse, TRP-1, and TRP-2 proteins increased by α-MSH.

Hwangchil leaf extract was found to have inhibitory activity simultaneously for four proteins, MITF, Tyrosinse, TRP-1, and TRP-2, depending on the concentration. Hwangchil leaf water extract was found to have a stronger inhibitory activity than the control drug Arbutin.

Experimental Example 6: Inhibitory effect on activation of MARKs induced by α-MSH

In order to know which signal proteins are involved in the melanin-producing signal pathway by the sample, phosphorylation of MARKs (ERK, JNK, p38) was analyzed by Western blot.

B16-F10 cells were dispensed in a 6-well plate so that the number of cells was 1.5 x 10 ⁵ cells / well and cultured for 24 hours. The culture medium was pre-treated with a sample (yellow pepper extract: 30, 100, 300 μg / mL) and a control drug (Arbutin: 2 mM) for 2 hours. Thereafter, α-MSH (200 nM) was treated to incubate for 24 hours. After the culture, the cells were washed once with DPBS, and a buffer added with a phosphatase inhibitor cocktail to a lysis buffer (25mM Tris pH 7.5 with H3PO4,2mM CDTA, 1% TritonX-100,2mM DTT, 10% glycerol) Cell lysates were prepared using.

The supernatant obtained by centrifuging the cell lysate at 13,000 rpm at 4 ° C for 10 minutes was used as a total protein extract. Proteins were quantified by Bradford analysis (Bio-Rad Laboratories, CA, USA). 20 μg of the quantified protein was boiled at 100 ° C. for 10 minutes, and then electrophoresed at 100 V on 10% SDS-PAGE.

Protein was attached to PVDF, blocked with 5% skim milk, and reacted with primary antibodies of ERK, p-ERK, JNK, p-JNK, p38, and p-p38, respectively. Subsequently, the secondary antibody HRP-conjugated anti-rabbit, anti-mouse antibody was treated.

After using ECL (enhancedchemiluminescence, Advansta Inc., CA, USA) as a substrate to identify the target protein, the membrane was developed using a C300 chemiluminescence imager (Azure Biosystems, Inc., CA, USA).

As shown in Figures 5a-5b, p-38 phosphorylation was decreased by treatment with hwangchilmul extract (Dm) in B-F10 cells induced by α-MSH. On the other hand, phosphorylation of ERK and JNK did not increase. These results indicate that the whitening activity is shown by controlling the phosphorylation of p38 of the upper protein of MITF.

Experimental Example 7: Hwangchil leaf extract (Dm) and p38 inhibitors have inhibitory effects on intracellular tyrosinase activation and melanin production

In order to investigate the effect of p38 signal transduction pathway as a mechanism for inhibiting melanin production of hwangchil leaf extract, an experiment was conducted using the p38 specific inhibitor SB203580.

B16-F10 cells were dispensed in a 6-well plate so that the cell number was 1 X 10 ⁵ cells / well and stabilized for 24 hours. After that, SB203580 (10 μM) was subjected to 1 hour pretreatment and then treated in the same manner as in the previous experiment. That is, after treating the hwangchil leaf extract 2 hours before, incubating with α-MSH (200 nM) for 72 hours, the intracellular tyrosinase and melanin contents were measured.

As shown in Figure 5c-5d, the group treated with α-MSH and SB203580 inhibited tyrosinase activation and melanin production compared to the group treated with α-MSH alone, and α-MSH, SB203580, and yellow leaf extract Comparing all treatments, tyrosinase activation and melanin production were significantly inhibited compared to the α-MSH and SB203580 treatment groups.

Experimental Example 8: Inhibitory effect on intracellular tyrosinase activation and melanin production of hwangchil leaf extract (Dm) and p38 inhibitor

In order to investigate the effect of p38 signal transduction pathway as a mechanism for inhibiting melanin production of hwangchil leaf extract, an experiment was conducted using the p38 specific inhibitor SB203580.

B16-F10 cells were dispensed in a 6-well plate so that the cell number was 1.5 X 10 ⁵ cells / well and stabilized for 24 hours. After that, SB203580 (10 μM) was pretreated for 1 hour and then treated in the same manner as in the previous experiment. That is, after treating the hwangchil leaf extract 2 hours before, incubating with α-MSH (200 nM) for 24 hours, the protein extract was measured for changes in MITF and tyrosinase proteins, which are melanin-related proteins, through Western blot analysis. .

As shown in Figure 5e-5f, the group treated with α-MSH and SB203580 decreased the amount of MITF and tyrosinase protein compared to the group treated with α-MSH alone, and both α-MSH, SB203580 and Hwangchilmul extract Compared with the treatment, the amount of MITF and tyrosinase protein was significantly reduced compared to the group treated with α-MSH and SB203580 together.

Experimental Example 9: Inhibition effect on intracellular tyrosinase activation and melanin production of a single component isolated from Hwangchil leaf extract (Dm)

The five single components isolated from the hwangchil leaf extract, linolenic acid, trilinolein, methyl chlorogenate, and the new substances DMW1 and DMW2, which inhibit melanin production, are shown in FIG. 6. Table.

As shown in Figures 7a-7c, the melanin content of the cells treated with α-MSH was increased compared to the control group not treated with α-MSH. New substances DMW1 and DMW2 dose-dependently inhibited intracellular tyrosinase activity and melanin production by α-MSH. New substance DMW1 and IC ₅₀ within the tyrosinase activity inhibition of cell DMW2 are each 3.232 μM and 97.390 μM, IC ₅₀ of the melanogenesis inhibition was good, each 1.221 μM and 3.426 μM significantly melanin production-inhibiting effect. Figure 7c is a photograph showing the color change of the pellet.

Experimental Example 10: Hwangchil tree leaf water extract (Dm) and gallium extract (Pf) alone or mixed treatment inhibitory effect of tyrosinase activation in cells

The hwangchil tree leaf extract (Dm) and gallium extract (Pf) were treated alone or mixed (Dm: Pf = 1: 1, 3: 1) to show the inhibitory effect on tyrosinase activity in cells. The method for measuring tyrosinase activity in cells is as described above.

Hwangchil tree leaf extract (Dm) and gallium extract (Pf) were treated with cells at 0.1 mg / ml (0.01%) or 0.3 mg / ml (0.03%).

When treated with α-MSH treated cells at a ratio of hwangchil: gallation = 1: 1 or 3: 1, the tyrosinase activity in the cell is further inhibited than when the hwangchil tree leaf extract and the gallium extract were treated alone. Did. In particular, the effect of the treatment in a 1: 1 ratio was the best.

Claims (5)

A composition for treating, improving or preventing skin pigmentation disease or symptom comprising at least one compound selected from the group consisting of compounds represented by the following formulas 1 and 2, and salts thereof:
[Formula 1]

; And
[Formula 2]

. A cosmetic composition for skin whitening comprising a compound represented by the following formulas 1 and 2, and one or more compounds selected from the group consisting of salts thereof:
[Formula 1]

; And
[Formula 2]

. A food composition for skin whitening comprising a compound represented by the following formulas 1 and 2, and at least one compound selected from the group consisting of salts thereof:
[Formula 1]

; And
[Formula 2]

. A compound represented by Formula 1 or salts thereof:
[Formula 1]

Method for separation of methyl (10E) -9,16-dihydroxyoctadeca-10,17-diene-12,14-diinoate compound comprising the following steps:
(a) extracting hwangchil tree leaves ( dendropanax morbifera lev . leaf) with a polar solvent;
(b) purifying only the extract soluble in the ethyl acetate solvent from the hwangchil tree leaf extract of step (a) to obtain an ethyl acetate extract fraction; And
(c) Purifying the ethyl acetate extract fraction to obtain methyl (10E) -9,16-dihydroxyoctadeca-10,17-diene-12,14-diinoate.

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