KR101308266B1 - Compositions for skin-whitening comprising widdrol - Google Patents

Abstract

The present invention is a cosmetic composition for whitening, including a whitening (widdrol), a food composition for whitening, a health functional food for the prevention or alleviation of pigmentation disease comprising the composition, and a pharmaceutical composition for the prevention or treatment of pigmentation disease It is about. The composition comprising the weedroll of the present invention has the advantage of no skin irritation and low cytotoxicity, but can inhibit tyrosinase activity associated with melanin production, thus preventing functional cosmetics for skin whitening, preventing skin pigmentation disease or It can be usefully used as a health functional food or a therapeutic agent for skin pigmentation disease that can be alleviated.

Description

Composition for skin-whitening comprising widdrol

The present invention relates to a method for using a widrol (meta) inhibits melanin production, and more specifically, a cosmetic composition for whitening, whitening food composition, including the above-mentioned weedrol prevention of pigmentation disease comprising the composition Or it relates to a palliative health functional food, and a pharmaceutical composition for the prevention or treatment of pigmentation diseases.

In general, with increasing environmental pollution, the preference for white skin is increased due to skin damage caused by ultraviolet rays, and as the pursuit of clear and clean skin with transparent makeup is increasing interest in how to obtain skin whitening effect. Human skin color is affected by the environment, race, and sex, but is generally determined by the brown melanin content. The melanin is a black pigment produced in the epidermis, which absorbs UV (ultraviolet) light energy of sunlight to protect cells deeper from UV damage, and is mainly produced in melanocytes. Such melanin should be produced at an appropriate level in the body. For example, when the melanin present in the base layer of the skin is excessively produced by various environmental factors such as ultraviolet rays and skin aging, skin pigmentation is increased, resulting in melasma and freckles. ), Side effects that cause seborrheic keratosis, etc. On the contrary, abnormally low production of melanin causes skin lesions such as vitiligo, and conversely, excessive synthesis of melanin by UV light not only damages the skin, Freckles are formed and from these lesions it can also cause skin cancer.

For the reasons described above, methods for minimizing the change in the production level of melanin due to external stimulation or intrinsic causes are sought from various angles, and melanin is mainly targeted to natural products so as not to affect the production level of melanin. Research is actively underway to develop a composition that can minimize the change in the level of production.

Conventionally, substances such as hydroquinone, ascorbic acid, kojic acid and glutathione have been used to improve skin hyperpigmentation or melanin overproduction. However, hydroquinone exhibits a predetermined whitening effect but severe skin irritation has a problem of limiting the blending amount to a very small amount. Ascorbic acid is easily oxidized, and the composition containing the same causes problems such as discoloration and discoloration. There was a disadvantage that can not be. In addition, kojic acid is unstable in solution due to the tendency of the formulation to change from yellow to brown over time due to photolysis progressing over time, which complicates the manufacturing process of the composition, and may cause mutagenesis, tumor promotion, and irritation. . In addition, thiol-based compounds such as glutathione and cysteine not only have a characteristic unpleasant odor, but also have problems with transdermal absorption, and glycosides and derivatives thereof have high polarity, making it difficult to use as a component of a cosmetic composition.

Therefore, the present inventors did not cause adverse effects on the human body, and as a result of diligent efforts to find a substance having excellent skin whitening effect, tyrosinase is superior to arbutin known as a whitening agent in the range in which widrol does not cause human toxicity. The present invention was completed by confirming the effect of inhibiting activity and inhibiting melanin production.

It is an object of the present invention to provide a cosmetic composition for whitening comprising a widrol.

Another object of the present invention is to provide a food composition for whitening comprising a weed roll.

Still another object of the present invention is to provide a health functional food for preventing or alleviating pigmentation disease including the food composition.

Another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of pigmentation diseases, including weedroll.

As one aspect for achieving the above object, the present invention provides a cosmetic composition for whitening comprising a widrol (widdrol).

As used herein, the term "widdrol" refers to a sesquiterpene compound as the compound of Formula 1. The general formula is represented by C ₁₅ H ₂₆ O and is named (7S, 9aS) -4,4,7,9a-tetramethyl-1,2,3,6,8,9-hexahydrobenzo [7] annulen-7- It is indicated by ol.

Weedroll of the present invention can be separated from natural substances, preferably from plants. Various organs, roots, stems, leaves, flowers, as well as plant tissue cultures of natural, hybrid, and mutant plants can be extracted and isolated. In addition, chemical synthesis is possible by methods known in the art, but those sold may be purchased and used.

The method for obtaining a weed roll from a plant is by various known extraction methods. For example, the plant is ground and then extracted with a lower alcohol solvent such as methanol, ethanol and the like, and then suspended in distilled water to give a nonpolar organic solvent such as normal hexane, ether, dichloromethane, chloroform, ethyl acetate. Or it can obtain by extracting and isolate | separating a nonpolar solvent soluble layer with these mixed solvents. From the obtained nonpolar organic solvent fraction, weedroll of formula (1) can be obtained. The extract can be further purified by performing one or more chromatography, wherein the type of column and the developing solvent can be controlled in various ways.

In an embodiment of the present invention, the juniper is crushed and then extracted with methanol, 1 to 100 times, preferably 3 to 8 times the volume of methanol 1 to 10 times, preferably 2 to 5 times repeatedly Water fractions and dichloromethane fractions were obtained by dividing with water and dichloromethane, respectively. The dichloromethane fractions were chromatographed to separate the weedrol.

In the present invention, the term "whitening" includes a method of increasing the brightness of the skin whose brightness has been reduced due to an excessive amount of pigment such as melanin, or maintaining the brightness of the skin at a constant level, skin having an increased brightness formed by the method, and the like. Can mean. For the purposes of the present invention, the whitening is carried out by means for inhibiting the synthesis of melanin formed in the skin by weedroll, but is not particularly limited thereto.

Melanin is a pigment component widely distributed in living organisms such as skin, hair, and eyes. It is synthesized in melanocytes in melanocytes of the human epidermal layer. Tyrosine is initiated by tyrosinase enzyme. Melanin is biosynthesized by oxidation and polymerization reactions converted to DOPA (3,4-dihydroxy-phenylalanine) or DOPA quinone. Biosynthesized melanin increases resistance to skin irritation such as ultraviolet rays, but measurement of tyrosinase activity is considered an important indicator of skin lightening effect because excessive melanin synthesis causes pigmentation such as spots, freckles and blotch. have.

In one embodiment of the present invention, weedroll significantly reduced melanin production induced by α-MSH within a range that does not cause toxicity to cells, and confirmed that it could inhibit the expression and activity of tyrosinase. It was.

Therefore, the composition including the weedroll of the present invention may have a function of helping to thin the color of the melanin pigment deposited on the skin, and prevents the excessive deposition of melanin pigment on the skin to prevent the occurrence of skin pigmentation disease By inhibiting it can have a function to help the skin whitening.

The skin pigmentation disease is caused by hyperpigmentation and tissue sclerotherapy after the use of drugs such as freckles, senile plaques, liver spots, blemishes, brown or dark spots, sunrays, cyanic melasma, and calcium antagonists. Hyperpigmentation, phototoxic reactions or other similar small fixed pigmented lesions after inflammation, wounds or dermatitis, including melanoma, scratches and burns, in sequelae, gravidic chloasma, oral contraceptives It may be included, but is not limited thereto.

The cosmetic composition of the present invention can be prepared into any of the formulations conventionally produced in the art and can be used as a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it may be prepared in the form of a flexible lotion, astringent lotion, nutrition lotion, nutrition cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder.

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

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as the carrier component. When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters. In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used. When the formulation of the present invention is a surfactant-containing cleansing, the carrier component is an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, isethionate, an imidazolinium derivative, a methyltaurate, a sarcosinate, a fatty acid amide ether. Sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

Compositions of the present invention include acute or chronic moisturizers (e.g. wetting agents, sealants, and agents that affect the natural moisturizing mechanism of the skin), antioxidants, sunscreens with UVA and / or UVB protection, softeners, anti-irritants , Vitamins, trace metals, antibacterial agents, vegetable extracts, perfumes, and / or other beneficial agents and compounds such as dyes and color components.

In addition, the composition of the present invention may include a surfactant. The surfactant can be an anionic surfactant, cationic surfactant, nonionic surfactant or compatible mixture of surfactants. The surfactant may also be an ampholytic or amphoteric surfactant having the properties of anions or cations depending on the pH of the composition.

The composition of the present invention may also comprise hydrotrope. Hydrotropes are compounds that have the ability to enhance the water solubility of other compounds. Specific examples of hydrotropes include, but are not limited to, sodium cumene sulfonate, ammonium cumene sulfonate, ammonium xylene sulfonate, potassium toluene sulfonate, sodium toluene sulfonate, sodium xylene sulfonate, toluene sulfonic acid, and xylene sulfonic acid. Other useful hydrotropes include sodium polynaphthalene sulfonate, sodium polystyrene sulfonate, sodium methyl naphthalene sulfonate, sodium camphor sulfonate, and disodium succinate.

Compositions of the present invention may also include thickening or gelling agents. Thickeners or gelling agents are, for example, polymers or copolymers unsaturated carboxylic acids or unsaturated esters, polysaccharide derivatives, gums, colloidal silicates, polyethylene glycols (PEG) and derivatives thereof, polyvinylpyrrolidone and derivatives thereof, polyacrylamides And derivatives thereof, polyacrylonitrile, hydrophilic silica gel or mixtures thereof.

In another aspect, the present invention provides a food composition for whitening comprising a weed roll.

The food composition for whitening may have a function of helping to thin the color of the melanin pigment deposited on the skin, to prevent the excessive deposition of melanin pigment on the skin to suppress the occurrence of skin pigmentation disease to whitening the skin It may have a helpful function.

The food composition for whitening of the present invention includes the form of pills, powders, granules, acupuncture, tablets, capsules or liquids, and the like to which the composition of the present invention can be added, for example, various foods, for example For example, there are drinks, gums, teas, vitamin complexes, and dietary supplements.

The food composition may be added to other ingredients that do not interfere with the whitening activity in addition to the weed roll, the kind is not particularly limited. For example, various herbal extracts, food acceptable food additives, natural carbohydrates, and the like may be contained as additional ingredients, such as conventional foods.

The term "food supplementary additive " in the present invention means a component which can be added to foods in a supplementary manner, and it can be appropriately selected and used by those skilled in the art as added to produce health functional foods of each formulation. Examples of food additives include flavors such as various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, colorants and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, Although pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like are included, the examples of the food additives of the present invention are not limited by the above examples.

Examples of such natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside, glycyrzin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.

In still another aspect, the present invention provides a health functional food for preventing or alleviating pigmentation disease of the food composition. In the present invention, the term "health functional food" refers to a food prepared and processed in the form of tablets, capsules, powders, granules, liquids and pills using raw materials or ingredients having useful functions for the human body. Here, the term "functionality" means that the structure and function of the human body are controlled to obtain nutritional effects or effects useful for health use such as physiological actions. The health functional food of the present invention can be prepared by a method commonly used in the art, and the preparation can be prepared by adding raw materials and ingredients commonly added in the art. In addition, unlike the general medicine has the advantage that there is no side effect that can occur when taking a long-term use of the drug with food as a raw material, can be excellent in portability.

The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). In general, the weedroll of the present invention in the preparation of food is added in an amount of 1 to 10% by weight, preferably 5 to 10% by weight in the raw material composition. However, in the case of long-term ingestion intended for health and hygiene purposes or for the purpose of controlling health, the amount can also be used in the above-mentioned range.

There is no particular limitation on the kind of the food. Examples of the food to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include healthy foods in a conventional sense.

In another aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of pigmentation diseases, including weedroll.

Preventing or treating skin pigmentation disease in the present invention is not only to prevent the deposition of melanin pigment on the skin by applying a composition comprising a weedroll to hyperpigmented skin, and furthermore to the melanin already deposited on the skin. Can mean any act of eliminating

The skin pigmentation disease is caused by hyperpigmentation and tissue sclerotherapy after the use of drugs such as freckles, senile plaques, liver spots, blemishes, brown or dark spots, sunrays, cyanic melasma, and calcium antagonists. Hyperpigmentation, phototoxic reactions or other similar small fixed pigmented lesions after inflammation, wounds or dermatitis, including melanoma, scratches and burns, in sequelae, gravidic chloasma, oral contraceptives It may be included, but is not limited thereto.

The pharmaceutical composition of the present invention may also be used as a single agent, and may be prepared and used as a complex preparation, further including a pharmaceutical composition known to have a recognized whitening effect.

The pharmaceutical composition of the present invention may be formulated in a pharmaceutical unit dosage form by adding a pharmaceutically acceptable carrier, excipient, or diluent.

By "pharmaceutically acceptable" is meant that it does not significantly irritate the organism and does not inhibit the biological activity and properties of the administered active substance.

The composition comprising a pharmaceutically acceptable carrier in the present invention may be a variety of oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups. In addition to the commonly used simple diluents, water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The pharmaceutical composition is any one selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquid solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories. It may have a formulation of.

In addition, in the composition of the present invention, weedroll may be included in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level refers to the type and severity of the subject, the severity, age, sex and activity of the drug. , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of the drug, and other factors well known in the medical arts. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. In consideration of all the above factors, it is important to administer an amount that can achieve the maximum effect with a minimum amount without side effects. Can be easily determined. Preferably in the present invention, the weedroll may be included in the composition at 0.001 to 200 μg / ml, more preferably at 0.001 to 100 μg / ml.

In another aspect, the present invention provides a method for preventing or treating skin pigmentation disorders by administering to a subject a pharmaceutical composition comprising Weedroll.

The subject is a subject in need of prevention or treatment of skin pigmentation disorders, which is a human, as well as cattle, horses, sheep, pigs, goats, camels, nutrition, dogs and cats that need treatment for skin pigmentation disorders and similar symptoms. It may be a mammal such as, but is not limited thereto.

As used herein, the term "administration" means introducing a composition of the present invention to an individual in any suitable manner, and the route of administration of the composition of the present invention may be administered via any general route as long as it can reach the desired tissue. have. The number of applications of the compositions of the present invention can be determined according to prescription, need or desire. The composition of the present invention may be prescribed in parallel with a surgical method such as a laser. It may also be incorporated into casts, bandages, dressings, gauze pads, and similar items.

The present invention includes administering a widroll in a pharmaceutically effective amount. It will be apparent to those skilled in the art that the appropriate total daily dose may be determined by the practitioner within the scope of sound medical judgment. In addition, it can be administered once or several times in divided doses. For purposes of the present invention, however, the specific therapeutically effective amount for a particular patient will depend upon the nature and extent of the reaction to be achieved, the particular composition, including whether or not other agents are used, the age, weight, Sex and diet of the patient, the time of administration, the route of administration and the rate of administration of the composition, the duration of the treatment, the drugs used or concurrently used with the specific composition, and similar factors well known in the medical arts.

The composition containing the widrol of the present invention has the advantage of no skin irritation and low cytotoxicity, but can inhibit tyrosinase activity related to melanin production, thus, functional cosmetics for skin whitening, skin pigmentation disease It can be usefully used as a health functional food or a therapeutic agent for skin pigmentation disease which can prevent or alleviate the disease.

1 is a schematic diagram illustrating a process for separating the weedroll and cedrol compound from the juniper extract.
Figure 2 is a graph showing the effect of the weedroll compound on the toxicity of B16F10 cells.
Figure 3 is a graph showing the effect of weedrol and cedrol on melanin production of cells induced by α-MSH (CR: cedrol, WR: weedroll, A: arbutin used as a positive control).
Figure 4 is a graph showing the effect of weedrol and cedrol on the tyrosinase activity of the cells induced by α-MSH (CR: cedrol, WR: weedroll, A: arbutin used as a positive control).
5 is a graph showing the effect of weedrol and cedrol on the expression level of tyrosinase protein induced by α-MSH in B16F10 cells (CR: cedrol, WR: weedroll, A: used as a positive control Arbutin).

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example 1 Preparation of Cedrol and Widrol

10 kg of juniper was measured and crushed into powder, and extracted with methanol. Specifically, five times the methanol solvent of the powder juniper sample was added and then extracted three times at 75 ℃. The extracted sample was filtered, concentrated in a vacuum condenser, and freeze-dried (FDU-2100, EYELA, Japan) to calculate the yield by weight method. As the solvent fraction, the concentrated solution concentrated under reduced pressure was separated into a water (H ₂ O) layer and a dichloromethane (CH ₂ Cl ₂ ) layer as shown in FIG. 1, and the water (H ₂ O) layer was again ethyl acetate, EtOAc) layer and water layer. The remaining water layer was further divided into an n- butanol (n- BuOH, n-butanol) layer and a water layer, and each fraction was concentrated under reduced pressure. Silicagel column chromatography was performed on the dichloromethane layer in the solvent fraction to separate cedrol and widrol (FIG. 1).

Example 1-1 content analysis of with rolls

The content of the weed roll separated from juniper by the extraction method was analyzed (Fig. 1).

As a result, as shown in Figure 1, 713.16g (yield: 7.136%) of the methanol extract was obtained, 399.36g of the dichloromethane fraction from the methanol extract, 15.88g of the separated weedroll of 0.1588 compared to 10 kg of juniper By weight, yield of 2.226% by weight compared to 713.16g of juniper methanol extract (the content of withol in the juniper extract is 2.226% by weight).

Example 1-2 Content Analysis of Cedrol

The content of cedrol separated from juniper by the extraction method was determined to be Cedrol by measuring EI-mass and NMR, to obtain 123.8 g of purely isolated cedrol (FIG. 1).

As a result, as shown in FIG. 1, 713.16 g of methanol extract (yield: 7.136%) was obtained, and 399.36 g of dichloromethane fraction was obtained from methanol extract, and 123.8 g of cedarol separated from 399.36 g of dichloromethane fraction was separated. The yield of 1.238% by weight compared to 10 kg of juniper, 17.36% by weight compared to methanol 713.16g of juniper extract (the content of cedrol in juniper extract is 17.36%).

As a result, it was confirmed that the weedroll in the juniper extract showed a very low content compared to the cedrol. These results suggest that weedroll is not the main component of the effect of the juniper extract itself.

Example 2 Analysis of Cytotoxicity between Cedrol and Weedroll

Mouse melanocytes (Mouse melanocyte) B16F10 was purchased from the American Type Tissue Collection (ATCC) and cultured in DMEM medium containing 10% fetal calf serum (FBS) and penicillin / streptomycin.

To determine the cytotoxicity of the sample before the whitening activity analysis and to determine the treatment concentration of the sample that does not induce cytotoxicity in subsequent experiments, the toxicity evaluation experiment was performed using the WST analysis (FIG. 2).

As a result, as shown in Fig. 2, both cedrol and weedroll did not show cytotoxicity by treatment up to 25 µg / ml. Based on the above results, the subsequent experiments were conducted to verify the whitening efficacy at 5-25 ㎍ / ㎖.

Example 3: Determination of melanin content in cells using B16F10 mouse melanoma cells

The mouse melanocytes B16F10 were treated with 200 nM of α-MSH to induce melanogenesis and analyzed for whitening activity by cedrol and withroll.

Specifically, 1x10 ⁵ B16F10 cells were dispensed in a D100 cell culture dish and cultured for one day, and after the sample processing, the cultured cells were washed with 1x phosphate buffered saline (PBS), followed by 10% dimethyl sulfoxide (dimethyl). 1 N sodium hydroxide (NaOH) containing sulfoxide, DMSO) was incubated at 80 ° C. for one hour, and then absorbance was measured at 475 nm using a microplate reader. Using the synthesized melanin solution, a standard curve was drawn and the measured values were substituted to calculate melanin production. As a positive control, arbutin, which is well known as a whitening active substance, was used, and the measured value was expressed as an average value of three repeated experiments (FIG. 3).

As a result, as shown in Figure 3, both cedrol and weedroll showed a concentration-dependent melanin production inhibition in the range of 5-25 ㎍ / ㎖ concentration, the activity was higher in the weedroll than cedrol. In addition, both substances showed higher activity at a lower concentration than arbutin used as a positive control group was confirmed to have a high whitening effect.

These results suggest that weedroll effectively inhibits melanin activity and shows whitening activity.

Example 4 Measurement of Tyrosinase Enzyme Activity of Cells Using B16F10 Mouse Melanoma Cells

After treating the cells with the sample in the same manner as in the measurement of melanin of Example 3, the cultured cells were washed with 1x PBS, followed by 1% (w / v) Triton X-100, 0.1 mM PMSF, 0.1 M phosphate buffer ( The cell lysate was prepared by treating the lysis buffer containing phosphate buffer, incubating for 30 minutes on ice, and centrifuging. After quantitating the protein by the Bradford method, 50 μg of protein was dispensed into 96-well plates, 2 mg / ml of L-DOPA was added, and then incubated at 405 nm for one hour every 10 minutes. The amount of dopachrome produced was measured (FIG. 4).

As a result, as shown in Figure 4, both cedrol and weedroll significantly reduced the tyrosinase enzyme activity of the cells induced by α-MSH, and these results were consistent with the previous melanin content. Moreover, weedroll inhibited tyrosinase enzyme activity more effectively.

Example 5 Analysis of Melanin Production-Induced Protein Expression Regulation of Cedrol and Weedroll

Protein expression changes of tyrosinase, a key enzyme that causes melanogenesis, were analyzed by western blot hybridization.

Specifically, the protein was extracted from the cultured cells treated with the sample, and the protein concentration was determined by Bradford assay, and then 50 ㎍ of the protein was electrophoresed with 10% SDS-PAGE, followed by a nitrocellulose membrane. After blotting, hybridization with the antibody of the protein of interest was performed. After washing the membrane and reacted with HRP tagged secondary antibody for one hour, protein expression was analyzed using a chemiluminescence detection system (FIG. 5).

As a result, as shown in Figure 5, it was shown that the tyrosinase protein expression induced by α-MSH was significantly reduced by the treatment of cedrol and withroll of 10 μg / ml, the extent of which It was higher in, similar to 100 ㎍ / mL arbutin used as a positive control. These results confirm that the anti-melanin production activity by cedrol and withroll is due to the inhibition of tyrosinase enzyme activity, a key protein of melanogenesis.

The above results suggest that weedroll of the present invention effectively inhibits tyrosinase enzyme expression and consequently inhibits melanin synthesis, thereby treating the disease caused by melanin deposition as well as the whitening effect.

Claims (6)

Whitening cosmetic composition comprising a widrol (widdrol).
The method of claim 1,
The composition comprises a formulation selected from the group consisting of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations and sprays. Cosmetic composition to have.
Whitening food composition comprising a weed roll.
Health functional food for the prevention or alleviation of pigmentation disease containing the composition of claim 3.
Pharmaceutical composition for the prevention or treatment of pigmentation diseases, including weedroll.
6. The method of claim 5,
The pigmentation disorders include freckles, senile plaques, liver spots, blemishes, brown or sunspots, sunrays, cyanic melasma, hyperpigmentation after drug use, gravidic chloasma, or scratches and burns. And hyperpigmentation following inflammation due to wounds or dermatitis.

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