KR100742267B1 - Extract of Chrysanthemum morifolium Ramat, the preparation method thereof and the cosmetic composition comprising the same for whitening - Google Patents

Abstract

The present invention relates to a whitening chrysanthemum extract, a preparation method thereof, and a whitening cosmetic composition containing the same as an active ingredient.

Seojang chrysanthemum extract of the present invention is excellent in melanin production inhibitory activity and no cytotoxicity, it can be usefully used in the cosmetic composition for whitening.

Description

Extract of Chrysanthemum Chrysanthemum, the preparation method thereof, and a whitening cosmetic composition containing the same as an active ingredient {Extract of Chrysanthemum morifolium Ramat, the preparation method according to the preparation and the same for whitening}

1 is a view showing the extraction, separation and purification process from Seojang chrysanthemum according to the present invention.

Figure 2 is a diagram showing the results of the analysis of the extraction fraction (1-4) of the ethyl acetate layer of the Seokjang Chrysanthemum extract according to the present invention.

Figure 3 is a diagram showing the melanin content when treated with a concentration (50, 100, 200㎛ / ㎖) seojang chrysanthemum extract (water layer, chloroform layer, ethyl acetate layer) according to the present invention.

Figure 4 is a diagram showing the melanin content when the extract fractions (1 ~ 4) of the ethyl acetate layer of seojang chrysanthemum extract according to the present invention by concentration (10, 50, 100㎍ / ㎖).

Figure 5 is a diagram showing the cytotoxicity test results of seojangchae chrysanthemum extract (water layer, chloroform layer, ethyl acetate layer) according to the present invention.

The present invention relates to an extract of Chrysanthemum morifolium Ramat, a preparation method thereof, and a cosmetic composition for whitening containing the same as an active ingredient.

Skin is a very important tissue that has direct contact with the external environment, protects the human body and has biochemical and physical functions. This tissue is largely divided into three parts: the epidermis, the dermis, and the hypodermis. Melanin is a phenolic polymer widely distributed in nature and is a black pigment present in the epidermal layer of the skin. It is made from melanocytes in the basal layer of the epidermis and migrates to epidermal cells called keratinocytes. There are no enzymes in the body that break down melanin, but they are removed by falling off the skin as keratinocytes fall off the epidermis.

Melanin absorbs the light energy of sunlight UV and protects the skin organs under the dermis from UV damage.It holds external harmful substances such as amines, free radicals, metal ions, harmful oxygen and free radicals generated in skin. Plays a useful role in protecting the skin from factors. In addition, the skin color of a person is determined by the amount of melanin in the skin cells. People with high melanin pigments have brown or black skin, while those with low melanin pigments have white skin.

When abnormally low melanin is produced, skin lesions such as vitiligo are induced, and conversely, if melanin is not synthesized excessively due to daylight, hormonal changes, inflammation, or drugs, or the keratinization is not performed smoothly, the skin is damaged completely. In addition to the skin pigments are deposited to form blemishes, freckles, and even from these lesions can cause skin cancer. Therefore, in order to prevent such pigmentation phenomenon, it is necessary to inhibit a part of melanin production process to reduce melanin production.

The biosynthesis of melanin is reported to be mainly due to tyrosinase action, but since the 1980s, DHICA (5,6-di) was induced by dopachrome tautomerase action in addition to the pathway of dopachrome conversion to DHI in vivo. A new pathway to convert to -hydroxyindole carboxylic acid has been identified. The synthesis of melanin from dopachrome via DHICA is believed to be a derivative route for cells to protect themselves from toxic production intermediate metabolites. The melanin thus produced is transferred to keratinocytes through the melanosomes, where it is lost to the surface of the skin along with the keratinization process for about 28 days.

The prevention of pigmentation on the skin has been mainly studied in the following four aspects. First, in order to regulate tyrosinase activity, the main enzyme of melanin synthesis, an inhibitor of tyrosinase synthesis or an antagonist of a substrate of tyrosinase is developed. Second, in order to reduce the function of melanocytes, the place of melanin biosynthesis in animals, a substance that is toxic to melanocytes is developed. Third, a dopa reducing material is developed to prevent oxidation of dopa, an intermediate metabolite of melanin synthesis pathway. Finally, the first enzyme tyrosinase, a melanogenesis mechanism and dopachrome tautomerase, a second enzyme catalyzing the conversion of dopachrome to DHICA, and indole-5, in DHICA Simultaneously reduces the activity of a third enzyme that catalyzes the conversion to 6-quinone-2-carboxylic acid.

Today, Asian women prefer white and clean skin like white jade and make it an important standard of beauty. Therefore, the development of whitening agent for treatment of abnormal skin pigmentation and satisfying beauty needs is being actively developed. Inhibition of melanin production to prevent skin blackening is the main purpose of whitening, melanin biosynthesis inhibitors have been applied to the treatment of skin diseases, food browning inhibitors or skin whitening agents in medicine, cosmetics and food.

At present, in the development of a whitening agent for skin beauty, a method of reducing the generated melanin pigment by decolorization and a method of inhibiting the activity of tyrosinase, an enzyme that forms melanin pigment, are known. However, whitening agents using tocopherols and vitamins used to reduce the melanin pigment are known to have a slight decolorizing effect of the melanin pigment. Therefore, attention is being paid to inhibitors that inhibit the production of melanin by inhibiting the activity of tyrosinase.

In the conventional cosmetic field, as a whitening component, for example, substances that inhibit tyrosinase enzyme activity such as kojic acid, arbutin, hydroquinone, vitamin C (L-Ascorbic acid) and these Derivatives of and various plant extracts have been used.

However, the stability in the prescription system is poor, the color is degraded, or the use thereof is limited due to the occurrence of off-flavor, efficacy at the biological level, unclear effect and safety problems. Kojic acid inhibits enzymatic activity by adsorbing copper ions present in the active site of tyrosinase, but has been discontinued as a raw material for cosmetics because of its instability, skin side effects and recent animal experiments. Vitamin C and its derivatives are difficult to use as raw materials for cosmetics due to their oxidative instability, and hydroquinone has excellent whitening effect on skin but allergic properties, toxicity to melanocytes, and permanent depigmentation to skin. It is highly irritating to, and is recently prescribed as a carcinogenic substance, so its use is banned and only a limited concentration is allowed in each country. In addition, arbutin is a derivative in which glucopyranoside is combined with hydroquinone, and has little side effects when using hydroquinone and inhibits the synthesis of melanin pigment without toxicity to the human body, thereby increasing melanin pigmentation. Although it has been proposed to be used as a therapeutic agent for skin diseases, it is disadvantageous to be partially degraded by enzymes present in the skin.

In addition to these whitening substances, research has been conducted to find whitening active ingredients in natural products, especially plants. Among them, white skin (Japanese Patent Application Laid-Open No. 55-44375, Small 64-26507, Small 64-83009, Pyeong 1-25687 No. 5-139950, Korean Patent Publication Nos. 92-002109, 97-021273, Licorice (Japanese Patent Laid-Open No. 60-214721, No. 60-214728, No. 63-23809, No. 64- 63506, Hei 1-149706, and Korean Patent Nos. 92-002109, 97-025601), Peony (Japanese Patent Laid-Open No. 61-246109 and Korean Laid-Open Patent No. 92-002111), Cinnamon Patent Nos. 63-30403, Hep. 5-139954), Gosam (Japanese Patent Laid-Open No. 64-26507 and Korean Patent Laid-Open No. 92-002110), Brown root (Japanese Laid-Open Patent Nos. 60-214727, Small 64-16709 ), Angelica (Japanese Patent Laid-Open No. 56-92211, Hei 4-26610), Mokdanpi (Japanese Laid-Open Patent No. 60-214721, No. 61-50915, No. 61-246109), Banha (Japanese Laid-Open Patent Publication) Pyeong 2-207028, Korea Publication No. 96-033442), aloe (Japanese Patent Laid-Open No. 52-44375, Hei 2-207030, Hei 5-139950) and many plant extracts and herbal extracts act on tyrosinase to produce melanin Although it has been found to suppress the, they also have a number of problems in using above the effective concentration in cosmetics or pharmaceuticals in terms of safety, discoloration potential. Therefore, much research is being conducted on the whitening cosmetics which are safe on skin and are less likely to be discolored.

On the other hand, chrysanthemum is a perennial herb belonging to the family Asteraceae. Chrysanthemum plants are known in the world about 200 species, chrysanthemum is known to lower blood pressure, prevent wind, blood circulation, lighten the body, ease the stomach, cold, headache, dizziness. In addition, Seojang chrysanthemum has the general characteristics of chrysanthemum, and because it has seven different colors, it is called 'Chrysanthemum'.

Thus, the inventors of the present invention, while researching among natural medicinal plants for whitening cosmetics that are safe to the skin and have little potential for discoloration, confirmed that melanin-producing inhibitory activity was excellent in seojang chrysanthemum extract native to Tibet, and completed the present invention.

The present invention is to provide a seojang chrysanthemum extract having a melanin production inhibitory activity.

In addition, the present invention is to provide a method for preparing seojang chrysanthemum extract.

In addition, the present invention is to provide a cosmetic composition for whitening containing seojang chrysanthemum extract as an active ingredient.

The present invention provides a seojang chrysanthemum extract having a melanin production inhibitory activity.

In addition, the present invention provides a method for preparing a seojang chrysanthemum extract,

(1) extracting by adding a solvent selected from water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof to Seojang Chrysanthemum,

(2) partition extraction of the extract obtained in step (1) with chloroform,

(3) partitioning and extracting the aqueous solution layer obtained in step (2) with ethyl acetate, and

(4) filtration, separation and purification of the ethyl acetate layer obtained in step (3).

Referring to the preparation method of the seojangchae chrysanthemum extract of the present invention in detail as follows.

Seojang chrysanthemum, native to Tibet, China, is washed with water to remove foreign substances, dried in the shade, and crushed. Appropriate amount of solvent is added to the pulverized Seojang Chrysanthemum so that it is completely immersed and extracted with stirring. As the extraction solvent, a solvent selected from water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof is preferable, and most preferably water. The amount of the extraction solvent is 10 to 30 times the dry weight of Seokjang Chrysanthemum, more preferably 15 to 25 times, and most preferably 20 times.

The extraction method can be impregnated or warmed at room temperature, preferably extracted at 70 ° C. for 3 hours.

The extract obtained above is cooled and filtered. The filtered solution is concentrated under reduced pressure using a rotary vacuum evaporator.

The concentrate and chloroform obtained above were put in a separatory funnel, sufficiently stirred, and then separated into an aqueous solution layer and a chloroform layer. At this time, preferably the amount of chloroform is to be the same amount as the concentrate. The aqueous solution layer and ethyl acetate obtained above were put in a separatory funnel, sufficiently stirred, and then separated into an aqueous solution layer and an ethyl acetate layer. At this time, preferably the amount of ethyl acetate is the same amount as the aqueous solution. The aqueous solution layer, chloroform layer, and ethyl acetate layer obtained above were evaporated and powdered by rotary evaporator, respectively, and the melanin production inhibitory activity using melanoma cells was confirmed to be the best in melanin production inhibitory activity in the ethyl acetate layer. .

The ethyl acetate layer of Seojang chrysanthemum extract obtained above was filtered, and then separated into four fractions (1 to 4) by chromatography using a preparative column. At this time, a mixed solvent of acetonitrile and water (60: 40, v / v%) is used as the mobile phase solvent. It was confirmed that the most melanin production inhibitory activity in fraction 2 of the fraction.

The present invention also provides a cosmetic composition for whitening containing the seojang chrysanthemum extract as an active ingredient.

The water, chloroform, and ethyl acetate layers of the extract of Seokjang Chrysanthemum according to the present invention reduce melanin production by B16 melanoma cells by up to about 18%, about 45%, and about 11.8%, respectively, compared to the arbutin treated group (FIG. 3). ). Fraction 2 detected between 20 to 50 minutes among fractions 1, 2, 3 and 4 obtained by chromatography of the ethyl acetate layer having the highest melanin production inhibitory effect among the solvent layers of the three extracts It reduces melanin production by 9.6% as compared to FIG. 4. In addition, each solvent layer shows no toxicity in cytotoxicity experiments conducted on B16 melanoma cells (FIG. 5).

Therefore, the melanin production inhibitory activity is excellent in the ethyl acetate layer and the ethyl acetate extract fraction 2 of the seojangchae chrysanthemum extract of the present invention and there is no cytotoxicity, it can be usefully used in the cosmetic composition for whitening.

Seojang chrysanthemum extract in the composition of the present invention is preferably contained in 0.005 to 20% by weight based on the total weight of the composition, but may be used in the above or below the range according to the intended use in the range that has a melanin inhibitory effect and does not appear toxic. Can be.

The composition of the present invention may further contain one or more active ingredients exhibiting the same or similar functions in addition to the seojang chrysanthemum extract.

Cosmetics prepared with a cosmetic composition containing the seojangchae chrysanthemum extract of the present invention as an active ingredient can be prepared in the form of a general emulsion formulation and solubilized formulation. Cosmetics of the emulsified formulations include nutrient cosmetics, creams, essences, etc., and cosmetics of the solubilized formulations are flexible cosmetics. In addition to the cosmetics containing the extract of the seojang chrysanthemum of the present invention, by containing a dermatologically acceptable medium or base may be prepared in the form of adjuvants that can be applied topically or systemically used commonly in the field of dermatology.

Suitable cosmetic formulations include, for example, emulsions, suspensions, microemulsions, microcapsules, microgranules or ionics (liposomes), nonionics obtained by dispersing an oil phase in a solution, gel, solid or pasty anhydrous product, aqueous phase. It may be provided in the form of a vesicle dispersant, in the form of a cream, skin, lotion, powder, ointment, spray or conceal stick. It may also be prepared in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

In addition, the cosmetic composition of the present invention, in addition to the seojang chrysanthemum extract, fatty substances, organic solvents, solubilizers, thickening and gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants Commonly used in water, ionic or nonionic emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or cosmetics It may contain adjuvants conventionally used in the cosmetic or dermatological fields, such as any other ingredients used. In addition, the above components may be introduced in an amount generally used in the dermatology field.

Examples of products to which the cosmetic composition of the present invention may be added include, for example, cosmetics such as astringent cosmetics, soft cosmetics, nourishing cosmetics, various creams, essences, packs, foundations, and the like, cleansing agents, soaps, treatments, and essences. Etc.

Specific formulations of the cosmetic composition of the present invention include skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisturizing lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, essence, nutrition essence, pack, Formulations such as soaps, shampoos, cleansing foams, cleansing lotions, cleansing creams, body lotions, body cleansers, emulsions, press powders, loose powders, eye shadows and the like.

Hereinafter, preferred examples and experimental examples are presented to help understand the present invention. However, the following Examples and Experimental Examples are provided only to more easily understand the present invention, and the contents of the present invention are not limited thereto.

Example 1 Preparation of Seojang Chrysanthemum Extract

1-1: Extracted from Seojang Chrysanthemum

10 g of Seokjang Chrysanthemum provided in Tibet, China was pulverized, and 200 ml of water was added thereto, followed by stirring and extraction at 70 ° C. for 3 hours. The extracted solution was cooled at room temperature for one day. The cooled solution was filtered through a 32 μm sieve and again filtered through filter paper. The filtrate was concentrated to 50 mL using a rotary decompression evaporator (Buchi Rotavapor R-200, Buchi Labotechuik AG. Switzerland). 50 ml and 50 ml of chloroform obtained above were placed in a separatory funnel, sufficiently stirred, and separated into an aqueous solution layer and a chloroform layer. In the next step, 50 ml of ethyl acetate was added to 50 ml of the aqueous solution layer obtained above, the mixture was sufficiently stirred, and the solution was separated into an aqueous layer and an ethyl acetate layer. The solutions of the aqueous solution layer, chloroform layer and ethyl acetate layer obtained above were evaporated to powder by rotary evaporation to give 850 mg of aqueous solution layer, 20 mg of chloroform layer and 138 mg of ethyl acetate layer, respectively.

1-2: Isolation and Purification of Seojang Chrysanthemum Extract

The ethyl acetate layer of the Seojang Chrysanthemum extract obtained in 1-1 was filtered using a 0.2 μm PTFE (polytetrafluoruethylene) syringe filter and used as a chromatography sample. 2 ml of the filtrate was poured into a reverse phase column (21.5 × 500 mm, GFC, C18, 13 μm, JAI, Japan) and mixed with acetonitrile: water = 60: 40 (v / v%) as a mobile phase solvent. Eluted with solvent. The elution rate of the mobile phase solvent was 3.5 ml / min and the detection wavelength was maintained at 254 nm.

The ethyl acetate layer was detected within 100 minutes of elution time and separated into four fractions (1-4). Each fraction was depressurized and concentrated using a vacuum concentrator, whereby fraction 1 between 3 and 18 minutes was 11.5 mg, fraction 2 between 20 and 50 minutes was 67.4 mg, fraction 3 between 50 and 58 minutes was 13.8 mg. And fraction 4 between 60 and 75 minutes yielded 14.1 mg.

Extraction, separation and purification process from Seojang chrysanthemum according to the present invention is shown in Figure 1, HPLC analysis results are shown in Figure 2 Shown in

Experimental Example 1 Whitening Effect of Seojang Chrysanthemum Extract

In order to determine the whitening effect of the seojangchae chrysanthemum extract of the present invention, the following experiment was carried out.

Melanocytes were used as the B16 melanoma cells (ATCC, CRL-6475) in the Pacific, and as culture medium, 10% (v / v%) FBS (fetal bovine serum), 1% antibiotic in RPMI 1640 medium. Made by the addition of (penicillin / streptomycin).

24 hours after inoculating B16 cells with 1 × 10 ⁵ cells / well as a standard, 50, 100, and 200 μg / ml of the seojang chrysanthemum extract prepared in Example 1 By concentration, the four extract fractions (1-4) of the ethyl acetate layer were treated with the concentrations of 10, 50 and 100 µg / ml and left to stand. After 48 hours, cells were transferred to a microcentrifuge tube, washed with PBS (phosphate buffer), and lysed in 250 μl NaOH containing 10% DMSO (dimethylsulfoxide). Lysed cells were left in an 85 ° C. incubator for 30 minutes to allow melanin to lyse. 200 μl of the solution was transferred to a 96 well plate and the absorbance was measured at 475 nm wavelength to detect melanin content. At this time, a group treated with arbutin, which is well known as a whitening ingredient, was used as a control.

The results are shown in FIGS. 3 and 4.

As shown in Figure 3, the melanin content of 38.75, 27.18, 11.8% at 50, 100, 200 ㎍ / ㎖ concentration of the ethyl acetate layer of the seojang chrysanthemum extract of the present invention showed a significantly lower melanin content than the control group Confirmed. In addition, it was confirmed that the ethyl acetate layer of the Seokjang Chrysanthemum extract of the present invention showed a lower melanin content than the water layer and the chloroform layer.

In addition, as shown in Figure 4, in the ethyl acetate extract fraction of the extract of Seokjang Chrysanthemum of the present invention, each concentration (10, 50 100 ㎍ / ㎖) appeared as 33.3, 26.5, 9.6%, respectively, melanin content Was found to be the lowest.

Therefore, the seojang chrysanthemum extract of the present invention is excellent in melanin production inhibitory activity, it can be usefully used in the cosmetic composition for whitening.

Experimental Example 2: Cytotoxicity Test of Seojang Chrysanthemum Extract

In order to determine the cytotoxicity of the extract of seojangchae chrysanthemum of the present invention, the following experiment was performed.

Cytotoxicity test was performed using MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, Sigma Chemical, USA). B16 melanoma cells were inoculated into 96 well plates at 2 × 10 4 cells / well, left for 2 days at 37 ° C. and 5% CO ₂ conditions, and then 100 μl of 5 mg / ml MTT solution was added to each well. It was left to stand for 4 hours more. Cells were then lysed in 100 μl DMSO and activity was detected by measuring absorbance at a wavelength of 570 nm using an ELISA reader.

The results are shown in FIG.

As shown in Figure 5, the seochae chrysanthemum extract of the present invention was shown to be non-toxic due to the high cell growth rate when compared to the control group not treated with any sample.

Examples of preparations for the compositions of the present invention are illustrated below.

Formulation Example 1 Flexible Lotion

Formulation example of the flexible lotion containing seojangchae chrysanthemum extract was prepared as shown in Table 1.

Raw material Content (% by weight) Seojang Chrysanthemum extract of the present invention 10.00 1,3-butylene glycol 1.00 Disodium ID 0.05 Allantoin 0.10 Dipotassium glycylizate 0.05 Citrix Acid 0.01 Sodium citrate 0.02 Glyceres-26 1.00 Arbutin 2.00 Hydrogenated Castor Oil 1.00 ethanol 30.00 Preservative a very small amount coloring agent a very small amount Flavor a very small amount Purified water Remaining amount

Formulation Example 2: Nutrition Cream

Formulation example of nutrition cream containing seojangchae chrysanthemum extract was prepared as shown in Table 2.

Raw material Content (% by weight) Seojang Chrysanthemum extract of the present invention 10.0 1,3-butylene glycol 7.0 glycerin 1.0 D-panthenol 0.1 Plant extracts 3.2 Magnesium Aluminum Silicate 0.3 PEG-40 Stearate 1.2 Stearic acid 2.0 Polysorbate 60 1.5 Lipophilic glyceryl stearate 2.0 Sorbitan sesquioleate 1.5 Cetearyl Alcohol 3.0 Mineral oil 4.0 Squalane 3.8 Carlylic / Capric Triglycerides 2.8 vegetable oil 1.8 Dimethicone 0.4 Dipotassium glycylizate a very small amount Allantoin a very small amount Sodium hyaluronate a very small amount Tocopheryl Acetate Quantity Triethanolamine Quantity Preservative Quantity Flavor Quantity Purified water Remaining amount

Seojang chrysanthemum extract of the present invention is excellent in melanin production inhibitory activity and no cytotoxicity, it can be usefully used in the cosmetic composition for whitening.

Claims (9)

(1) Chrysanthemum morifolium Ramat is extracted by adding a solvent selected from water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, (2) partitioning and extracting the crude extract obtained in step (1) with chloroform to extract and extract the aqueous layer obtained with ethyl acetate, (3) HPLC of the ethyl acetate layer obtained in step (2) under a column size of 21.5 X 500 mm, acetonitrile and water mixed with 60:40 (v / v%) as a mobile phase under elution rate of 3.5 ml / min Whitening cosmetic composition containing the fraction eluted between 20 minutes to 50 minutes as an active ingredient. The cosmetic composition for whitening according to claim 1, wherein the solvent of step (1) is water. The cosmetic composition for whitening according to claim 1, wherein the chloroform or ethyl acetate of step (2) is added in the same amount to the aqueous solution layer, respectively. delete delete delete delete delete delete

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