KR101742167B1 - Skin-lightening Composition Using an Extract of Dipterocarpus intricatus - Google Patents

Abstract

The present invention relates to a skin whitening treatment using Dipterocarpus intricactus extract exhibiting a high tyrosinase inhibitory activity and exhibiting a melanin inhibitory activity in a concentration-dependent manner when treated with melanoma cell line B16F10 stimulated with alpha-MSH stimulating melanin stimulating agent ≪ / RTI > The Dipterocarpus intricactus extract is similar to arbutin, an inhibitor of tyrosinase, which is one of the synthetic whitening components, and has a markedly higher melanin production inhibitory activity than arbutin, a synthetic whitening agent in functional cosmetics.

Description

[0001] The present invention relates to a composition for skin whitening using an extract of Dipterocarpus intrica tussa (Dipterocarpus intricatus)

The present invention relates to the use of Dipterocarpus < RTI ID = 0.0 > The present invention relates to a composition for skin whitening using an extract of the present invention.

As the development of medical technology and improvement of living standards are strengthening the desire for beauty of the people and the perception that the appearance itself is a social competitiveness is increasing, the preference for skin whitening is increasing and the cosmetics market is expanding (J. Korean Soc. Food Sci. Nutr., 42 (3): 355 (2013)).

The color of a person's skin depends on various factors, especially season, race and sex, and is mainly determined by the amount of melanin, carotene, and hemoglobin, the latter being the most crucial factor.

Melanin is a phenolic polymer natural pigment widely found in living organisms. It is synthesized in melanosomes in melanocytes in the basal layer of the epidermis and transformed into peripheral keratinocytes to show human skin color. When melanin is abnormally low, skin lesions such as vitiligo are induced. On the contrary, when excessive production is produced, acquired hyperpigmentation such as melasma, postinflammatory melanoderma, solar lentigo, , ≪ / RTI >

Melanin is synthesized by the continuous oxidation of tyrosine (tyrosine), specifically tyrosinase, tyrosinase related protein-1 (TRP-1), and tyrosinase related protein-2 (DOPA, 3,4-dihydroxyphenylalanine) and converted to dopaquinone (DOPA quinone) by non-enzymatic reaction, spontaneous oxidation and synthesis by amino acid or protein polymerization (Korean J. FOOD SCI. TECHNOL., 32 (3): 736, 2000; Journal of Life Science, 23 (12): 1445, 2013)).

Since tyrosinase plays the most important role as the first step in the production of melanin, the search for a variety of tyrosinase inhibitors has continued to be made for the development of whitening agents (Kor. J. Pharmacogn., 44 (3): 220, 2013). Recently, research on suppression of melanin synthesis targeting signaling substances such as MITF (microphtalmia associated transcription factor) has been actively conducted. The major intracellular signaling pathway is the cyclic monophosphate / protein kinase A pathway, which stimulates the expression of MITF, a transcriptional regulator, via PKA, CREB1 (cAMP responsive element binding protein 1) (3): 289, 2013; J. Oriental Physiology & Pathology, 24 (2): 296 (1986), which promotes transcription of tyrosinase, TRP-1 and TRP-2 , 2010).

Currently, arbutin, the most commonly used whitening agent, is an inhibitor that competes with L-tyrosine, and kojic acid chelates the tyrosinase active site, resulting in tyrosine-dopa, dopaquinone (Korean J. Plant Res., 26 (5): 526, 2013; J. Korean Soc. Food Sci. Nutr., 41 (2): 156, 2012). Arbutin and kojic acid are known to have powerful whitening effect, but studies have been actively made to find substances having whitening activity from natural substances with relatively few side effects due to some side effects such as skin irritation.

The present invention discloses Dipterocarpus intricactus extract having tyrosinase inhibitory activity and melanin production inhibitory activity.

It is an object of the present invention to provide a skin whitening composition using Dipterocarpus intricactus extract.

Other and further objects of the present invention will be described below.

The present invention is based on the finding that the Dipterocarpus intricactus extract exhibits high tyrosinase inhibitory activity and is treated with Melanoma cells B16F10 stimulated with alpha-MSH, a melanogenesis stimulant, as shown in the following Examples and Experimental Examples , And further confirmed that tyrosinase inhibitory activity or melanin production inhibitory activity is equal to or superior to arbutin, which is a synthetic whitening agent. Dipterocarpus intricatus is a plant belonging to Dipterocarpaceae family, which is native to Thailand, Cambodia, Laos and Vietnam.

The present invention provides skin whitening compositions of the present invention, which are based on the results of these experiments, characterized in that they contain Dipterocarpus intricactus extract as an active ingredient.

In the present specification, the term "Dipterocarpus intricatus extract" refers to an extract of Dipterocarpus intrica tussa stem, leaf, fruit, flower, root or the like to be extracted with water, a lower alcohol having 1 to 4 carbon atoms such as methanol, ethanol, (DMF), dimethylsulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or a mixture thereof. The solvent is preferably selected from the group consisting of methylene chloride, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, Refers to an extract obtained by leaching using a mixed solvent, an extract obtained by using a supercritical extraction solvent such as carbon dioxide or pentane, or a fraction obtained by fractionating the extract, and the extraction method includes considering the polarity, extraction degree, and preservation degree of the active substance Any method such as cooling, reflux, heating, ultrasonic irradiation, supercritical extraction, and the like can be applied. In the case of the fractionated extract, the fraction is suspended in a specific solvent and then mixed with a solvent having a different polarity. The extract is adsorbed on a column packed with silica gel or the like, and then a hydrophobic solvent, a hydrophilic solvent, Quot; means fractions obtained as a mobile phase. Also, the meaning of the extract includes a concentrated liquid extract or a solid extract in which the extraction solvent is removed by a method such as freeze drying, vacuum drying, hot air drying, spray drying and the like. Preferably an extract obtained by using water, ethanol or a mixed solvent thereof as an extraction solvent, and more preferably an extract obtained by using a mixed solvent of water and ethanol as an extraction solvent.

In the present specification, the term "active ingredient" alone means an ingredient which exhibits the desired activity or can exhibit activity together with a carrier which is not itself active.

In the present specification, "skin whitening" can be understood as an increase in skin brightness which is reduced due to excessive production of melanin. Specifically, skin whitening can be improved by improving the skin, freckles, Treatment, prevention, suppression of expression or delay.

The composition for skin whitening of the present invention may further comprise any compound or plant extract known to have safety and skin whitening activity for the purpose of increasing or reinforcing the skin whitening effect, in addition to the effective ingredient. Specific examples of such compounds or extracts include albutin, niacinamide, ascorbyl glucoside, alpha-bisabolol, and oil soluble licorice (Glycyrrhiza) extract. Other known whitening ingredients include cosmetics (Functional Cosmetics Standards and Test Methods) issued by the Ministry of Health, Labor and Welfare. Such compounds or plant extracts may be included in the skin whitening composition of the present invention in combination with one or more thereof.

In the skin whitening composition of the present invention, the active ingredient may be contained in any amount within a range that does not toxicity to the human body according to the purpose of use, formulation, etc. Specifically, it is preferably 0.01% by weight to 99.99% % ≪ / RTI > by weight.

The skin whitening composition of the present invention can be identified as a cosmetic composition in a specific embodiment.

The cosmetic composition of the present invention may contain, in addition to its active ingredient, conventional additives such as stabilizers, solubilizing agents, surfactants, vitamins, colorants and antioxidants, and carriers commonly used in cosmetic compositions.

The cosmetic composition of the present invention may be prepared in any formulations conventionally produced in the art and may be in the form of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, , Oil, powder foundation, emulsion foundation, wax foundation, spray, and the like. In a specific product form, it may be formulated into a formulation of a flexible lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

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

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In the case of a spray, in particular, / Propane or dimethyl ether.

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

When the formulation of the present invention is a suspension, a carrier, such as water, a liquid diluent such as ethanol or propylene glycol, a suspension such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar, etc. may be used.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component is selected from aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, 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.

The cosmetic composition of the present invention may be prepared by adding a skin wrinkle-improving ingredient, an ultraviolet ray-protecting ingredient or the like known in the art in order to reinforce or add a skin-related activity. Specific examples of such components may refer to the functional cosmetic products according to the Cosmetic Act.

The cosmetic composition of the present invention can be produced according to a method for producing a cosmetic composition which is usually carried out in the art, except that it contains an active ingredient showing skin whitening activity.

The skin whitening composition of the present invention can be grasped as a food composition in another specific embodiment.

The food composition of the present invention can be manufactured as health functional foods such as functional beverages, health supplements, special nutrition supplements, etc. The forms of foods include beverages such as tea, juice, carbonated beverage, ionic drink, milk, Food products such as processed oil, gum, rice cake, Korean food, bread, confectionery and cotton, tablets, capsules, rings, granules, liquid, powder, flakes, paste, syrup, gel, jelly and bar And so on.

The food composition of the present invention may contain sweetening agents, flavoring agents, physiologically active ingredients, minerals and the like in addition to the active ingredients thereof.

Sweetening agents may be used in an amount that sweetens the food in a suitable manner, and may be natural or synthetic. Preferably, natural sweeteners are used. Examples of natural sweeteners include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose and maltose.

Flavors may be used to enhance taste or flavor, both natural and synthetic. Preferably, a natural one is used. When using natural ones, the purpose of nutritional fortification can be performed in addition to the flavor. Examples of natural flavoring agents include those obtained from apples, lemons, citrus fruits, grapes, strawberries, peaches, and the like, or those obtained from green tea leaves, Asiatica, Daegu, Cinnamon, Chrysanthemum leaves and Jasmine. It may also be obtained from ginseng (red ginseng), bamboo shoots, aloe vera, banks and the like. The natural flavoring agent may be a liquid concentrate or a solid form of extract. Synthetic flavors may be used depending on the case, and synthetic flavors such as esters, alcohols, aldehydes, terpenes and the like may be used.

Examples of the physiologically active substance include catechins such as catechin, epicatechin, gallocatechin and epigallocatechin, and vitamins such as retinol, ascorbic acid, tocopherol, calciferol, thiamine and riboflavin.

As the mineral, calcium, magnesium, chromium, cobalt, copper, fluoride, germanium, iodine, iron, lithium, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, silicon, sodium, sulfur, vanadium and zinc can be used.

In addition, the food composition of the present invention may contain preservatives, emulsifiers, acidifiers, thickeners and the like as needed in addition to the above sweeteners.

It is preferable that such preservatives, emulsifiers and the like are added in a small amount so long as they can attain the use to which they are added. Trace amounts, when expressed numerically, range from 0.005% to about 0.5% by weight, based on the total weight of the food composition.

Preservatives that can be used include calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, and EDTA (ethylenediaminetetraacetic acid).

Examples of the emulsifier which can be used include acacia gum, carboxymethyl cellulose, xanthan gum, pectin and the like.

Examples of the acidulant that can be used include acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, and phosphoric acid. Such an acidulant may be added so that the food composition has a proper acidity for the purpose of inhibiting the growth of microorganisms other than the purpose of enhancing the taste.

Agents that may be used include suspending agents, sedimentation agents, gel formers, bulking agents and the like.

Food additives and food additives according to the Food Sanitation Law may be referred to in relation to additives and other additives that are required to be used depending on the type of food or type of food.

The skin whitening composition of the present invention can be identified as a pharmaceutical composition in a specific embodiment.

When the composition for skin whitening of the present invention is identified as a pharmaceutical composition, the target disease can be understood as skin hypercholesterolemia. Specific examples of such hypercholesterolaemia include freckles, senile spots, liver, spots, brown or black spots, Hyperglycemia after drug use such as cyanic melasma, calcium antagonist, sequela due to sclerotherapy, gravidic chloasma, mellitus in women taking oral contraceptives, But are not limited to, post-inflammatory hyperpigmentation due to scratches or dermatitis, including scratches and burns, phototoxic response or other similar small fixed pigmented lesions.

The pharmaceutical composition of the present invention may be prepared into oral formulations or parenteral formulations according to the route of administration by conventional methods known in the art, including pharmaceutically acceptable carriers in addition to the active ingredient. The term "pharmaceutically acceptable" as used herein means that the application (prescribing) subject does not have the above-mentioned toxicity that is adaptable without inhibiting the activity of the active ingredient.

When the pharmaceutical composition of the present invention is prepared into an oral formulation, it may be formulated into powder, granules, tablets, pills, sugar tablets, capsules, solutions, gels, syrups, suspensions, wafers And the like. Examples of suitable pharmaceutically acceptable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, xylitol, starch such as corn starch, potato starch and wheat starch, cellulose, methylcellulose, ethylcellulose, Cellulose derivatives such as sodium carboxymethyl cellulose and hydroxypropylmethyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, And the like. In case of formulation, a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, and / or an excipient may be formulated according to need.

When the pharmaceutical composition of the present invention is prepared into a parenteral dosage form, it may be formulated in the form of an injection, transdermal drug delivery, nasal aspirate and suppository together with a suitable carrier according to methods known in the art. Examples of suitable carrier in the case of injection preparation include sterilized water, polyol such as ethanol, glycerol or propylene glycol, or a mixture thereof. Preferably, the carrier is selected from the group consisting of Ringer's solution, phosphate buffered saline containing triethanolamine, , 5% dextrose, and the like may be used. When formulated with a transdermal drug, it can be formulated in the form of an ointment, a cream, a lotion, a gel, a solution for external use, a pasta, a liniment, or an air-roll. The nasal inhalant may be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc. The suppositories may be witepsol, polyethylene glycol, cacao butter, laurin, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters and the like can be used.

The formulation of pharmaceutical compositions is well known in the art and can be specifically described in Remington ' s Pharmaceutical Sciences (19th ed., 1995). This document is considered part of this specification.

The preferred dosage of the pharmaceutical composition of the present invention is 0.001 mg / kg to 10 g / kg per day, preferably 0.001 mg / kg to 1 g / day, depending on the patient's condition, body weight, sex, age, / kg < / RTI > The administration can be carried out once or several times a day. Such dosages should in no way be construed as limiting the scope of the invention.

As described above, according to the present invention, it is possible to provide a skin whitening composition using Dipterocarpus intrica tussa extract.

The active ingredient, Dipterocarpus intricactus extract, of the present invention is similar to arbutin, an inhibitory activity of tyrosinase, which is one of the synthetic whitening components, and has a markedly higher inhibitory activity on melanin production than arbutin.

The skin whitening composition of the present invention can be produced into foods, cosmetics, medicines, and the like, and the use of skin whitening substances derived from natural substances may have relatively few side effects as compared with synthetic skin whitening agents.

Fig. 1 shows the results of inhibiting the melanin production of Dipterocarpus intricactus extract in B16F10 melanoma cells stimulated with α-MSH, which is a melanin-forming stimulant.

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these examples and experimental examples.

< Example > Dipterocarpus Intricatus  Preparation of extract

Dried Dipterocarpus &lt; RTI ID = 0.0 &gt; intricatus ) 1 g of 70% ethanol was added to 100 g of leaf, repeatedly extracted once at room temperature for 24 hours, and then filtered with a filter paper. The resulting 70% ethanol filtrate was concentrated under reduced pressure and then lyophilized to obtain a Dipterocarpus intrica tus extract.

< Experimental Example > Whitening activity experiment

<Experimental Example 1> Tyrosinase inhibitory activity test

20 μl of the sample (NIBR No. 115) diluted by concentration was added to 220 μl of 0.1 M potassium phosphate buffer (pH 6.5), and then 20 μl of 1250 unit / ml mushroom tyrosinase was added. After incubation at 37 ° C for 10 minutes, the absorbance was measured using an ELISA reader at 490nm, and the inhibition rate according to the treatment concentration of the sample was determined according to the following equation, and IC ₅₀ The values are shown in Table 1 below. Arbutin was used as a positive control.

Inhibition% = 100- (A-B) / (C-D) x100

A: Absorbance after reaction of the sample solution

B: absorbance of the sample solution measured by replacing with tyrosinase buffer

C: absorbance after the reaction of the blank

D: absorbance of the blank sample measured by replacing tyrosinase with buffer

Inhibition rate of tyrosinase Sample IC ₅₀ ([mu] g / ml) NIBR No. 115 141.46 Arbutin 136.77

As can be seen in Table 1, the absorbance of melanin produced by oxidation of L-tyrosine substrate and enzyme mushroom tyrosinase was measured to determine the inhibitory activity of tyrosinase, an enzyme that plays an important role in melanin production As a result of the measurement, the sample of the example shows a tyrosinase inhibitory activity similar to arbutin, which is a whitening agent in the functional cosmetics.

<Experiment 2> Melanin formation inhibitory activity experiment

<Experimental Example 2-1> Cell culture

B16 / F10 cells were purchased from the Korean Cell Line Bank (KCLB) as a melanoma cell line in rats, and 100 units / ml penicillin-streptomycin (GIBCO, Grand Island, NY, USA) and 10% fetal bovine serum Were cultured in a 5% CO ₂ incubator at 37 ° C using RPMI 1640 or DMEM medium (GIBCO, Grand Island, NY, USA) containing 1% Respectively.

<Experimental Example 2-2> Evaluation of melanin formation inhibitory activity

B16F10 melanoma cell line was divided into 6 well plates at 1 × 10 ⁵ cells / well and after 24 h, 100 nM α-melanocyte stimulating hormone (α-MSH) and a positive control group And cultured for 72 hours. After washing with PBS 2 times, lysis buffer (100 mM sodium phosphate (pH 6.8), 0.1 mM PMSF, 1% Triton X-100) was added and dissolved at -80 ° C for 30 minutes. After the cell harvesting, 1N NaOH containing 10% DMSO was reacted at 65 ° C for 1 hour to dissolve the pellet, and the absorbance was measured at 405 nm.

The results are shown in Fig.

Referring to FIG. 1, it can be seen that the samples of the examples show melanin production inhibitory activity in proportion to the treatment concentration, and the melanin production inhibitory activity is remarkably superior to that of the positive control group, arbutin.

<Experimental Example 2-3> Cytotoxicity evaluation - MTT Assay

B16F10 cells were plated at 1 × 10 ⁴ cells / well in a 96-well plate and the samples were treated with concentrations (62.5, 125, 250, 500 ㎍ / ml) and cultured for 24 hours. The medium containing 5 mg / ml MTT solution was added and incubated for 2 hours. MTT reagent was removed, and DMSO was added to dissolve formazan. The absorbance was then measured at 540 nm using an ELISA reader.

The results of treatment concentration are shown in Fig. 2, and the IC _{50 values} are shown in Table 2 below.

MTT Assay Results Sample IC ₅₀ ([mu] g / ml) NIBR No. 115 > 500

The results are shown in Table 2. The results show that the IC ₅₀ of the samples of the examples was 500 μg / ml or more, indicating that the result of inhibiting the melanin formation is not the result of cytotoxicity, and the lowest concentration Lt; RTI ID = 0.0 &gt; g / ml &lt; / RTI &gt;

Claims (5)

A composition for skin whitening comprising an extract of Dipterocarpus intricactus leaf as an active ingredient.
The method according to claim 1,
Wherein the extract of Dipterocarpus intrica tus leaves is obtained by using water, ethanol or a mixed solvent thereof as an extraction solvent.
3. The method according to claim 1 or 2,
Wherein the composition is a cosmetic composition.
delete An extract containing Dipterocarpus intricactus as an active ingredient,
Wherein said Dipterocarpus intrica tussa extract is obtained by extracting Dipterocarpus intrica tussa leaf and using water, ethanol or a mixed solvent thereof as an extraction solvent. Composition.

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