KR20050096556A - Skin whitening ingredients containing astaxanthin - Google Patents

Abstract

The present invention relates to a skin whitening agent containing astaxanthin that inhibits the activity of melanin-forming tyrosinase, and more particularly, using natural astaxanthin extracted from Hamatococcus pluvialis . The present invention relates to a cosmetic for skin whitening, a food composition and an agent for inhibiting activity of tyrosinase.

Description

Skin whitening agent containing astaxanthin {SKIN WHITENING INGREDIENTS CONTAINING ASTAXANTHIN}

The present invention relates to a skin whitening agent containing astaxanthin that inhibits the activity of tyrosinase forming melanin, and more specifically, to be used as a component of cosmetics and pharmaceuticals for skin whitening and food composition for skin whitening. Astaxanthin-containing skin lightening agent that can be.

The most important factor in determining the color of human skin is melanin, which is determined by the amount, nature and distribution of melanin. Melanin is a phenolic polymer that is widely distributed in nature. It is a complex of black pigment and protein. Melanin is mainly present in the skin of the human body and functions to protect the body from ultraviolet rays. High levels of melanin mean that you have an effective response system to protect your skin from physical or chemical toxicants.

However, when excessively present in melanin in the skin tissue, the over-produced melanin forms pigmentation, spots, freckles and promotes skin aging. In the past, natural products such as yulmu and cucumber have been used for skin whitening in connection with skin whitening, but these natural products have not been related to the overproduction of melanin.

In recent years, melanin is formed as a chemical pathway through which melanin is produced through tyrosinase (tyrosine) through tyrosinase (DOPA) and dopaquinone (DOPA-quinone). Many of the pathways, such as melanocytes, which migrate from melanocytes to keratinocytes, produce melanin.

In recent years, skin whitening by inhibiting melanin production includes a method of blocking ultraviolet rays, inhibiting the synthesis of core carbohydrates necessary for the activity of tyrosinase, and inhibiting the activity of tyrosinase, an enzyme related to melanin formation. A method, a method of preventing the division of melanocytes using a substance having specific toxicity to melanocytes, a method of using vitamin C derivatives and placental extracts, and the like have been disclosed.

In Japanese Patent Laid-Open No. 6-192062, hydroquinone has been disclosed as a whitening substance, but the hydroquinone has an excellent whitening effect, but there is an inadequate problem in use as a material such as cosmetics as a carcinogenic substance.

In Japanese Patent Laid-Open No. 56-7710, kojic acid has been disclosed as a whitening substance. Kojic acid has an excellent whitening effect due to its excellent inhibitory ability of tyrosinase, but since the problem of toxicity of kojic acid has been discussed, cosmetics, food There is a problem that it is not suitable for use as a material such as.

Japanese Patent Application Laid-Open No. 4-9315 discloses arbutin, which is extracted from bilberry inhabiting alpine areas as a natural plant as a whitening substance or obtained through synthesis, but has been pointed out by skin irritation.

On the other hand, astaxanthin is a carotenoid-based pigment obtained from aquatic animals, including shellfish such as shrimp and crabs. Astaxanthin is known to have antioxidant effects (Kurashige et al) and various cancer prevention effects. Carcinogenesis 15: 15-19), immune effect (Nutr. Cancer 23: 171-183) and the like have been known to have the effect, but is not yet known with respect to skin whitening.

In the health food market, the importance of skin care-related functional products is increasing more and more, the consumer's desire for products is increasingly fragmented, and the actual whitening effect is greatly required. In this regard, almost all conventional cosmetic companies have developed a whitening agent to satisfy the needs of these consumers, but these studies have focused on the efficacy of applying the skin, and few products satisfying the whitening effect of consumers by ingestion have been developed. It is our present situation that no.

In fact, the raw materials reported to the academic community as whitening ingredients through ingestion are very small, such as batamine C, vitamin E, guava extract, and cosmetics containing hydroquinone, kojic acid, and albumin applied to the skin. Although the compounds have been tested for skin whitening efficacy through various in vitro tests, many derivatives are still being synthesized or searched through natural products for reasons such as the above, which are not satisfactory. The development of whitening products through administration is currently a long time.

The present inventors completed the present invention for the first time by confirming that astaxanthin, particularly astaxanthin extracted from hematococcus, inhibits the activity of tyrosinase that mediates melanin synthesis, thereby effectively inhibiting melanin production. Was done.

It is an object of the present invention to provide a skin whitening agent for inhibiting tyrosinase activity containing astaxanthin as an active ingredient and a food composition using the same.

The present invention provides a skin whitening agent for inhibiting tyrosinase activity containing astaxanthin component of Formula 1 as an active ingredient.

[Formula 1]

The present invention also provides a cosmetic composition for skin whitening containing the astaxanthin component.

The present invention also provides a health functional food for skin whitening further comprising a food additive that is acceptable as the astaxanthin component and a food additive.

The present invention also provides a pharmaceutical composition for inhibiting tyrosinase activity further comprising the astaxanthin component and a pharmaceutically acceptable carrier.

The present invention relates to a skin whitening agent, a cosmetic composition, a dietary supplement, and a pharmaceutical composition using astaxanthin, and this effect is due to the excellent antioxidant activity of astaxanthin, and astaxanthin inhibits the activity of tyrosinase. It is believed that the effect of skin whitening is expressed by acting to inhibit the production of melanin.

Astaxanthin (3,3'-dihydroxy-β, β-carotene-4,4'-dione) component of the present invention occupies most of the carotenoid pigments contained in crustaceans, Xanthophyll of carotenoids Red pigment belongs to Astaxanthin is a natural krill, lobster, shrimp, crab, salmon, yeast such as Phaffia rhodozyma , and Acetabularia mediterranea , Chlamydomonas nivalis . Algae such as Chlamydomonas nivali s), Euglena rubida , and Haematococcus species , but especially Haematococcus pluvialus flotow and Hematococcus raku . It is desirable to obtain from the striae ( Haematococcus lacustris ). In addition, commercially available astaxanthin from MPT-Korea may be used.

As a method for obtaining astaxanthin from Haematococcus species among the strains, incubating hematococcus under a supply of carbon dioxide and a light source in an incubator, suspending it in water and microwave treatment, It is preferable to obtain astaxanthin by extracting the alcohol with a solvent and then concentrating under reduced pressure.

According to a preferred embodiment of the present invention, the medium and growth conditions for the culture of hematococcus are as follows.

First, as a component of the medium in deionized sterilized water for culturing hematococcus, sodium acetate, L-asparagine, yeast powder, MgCl ₂ and 6H ₂ O), FeSO ₄ · 7H ₂ O, CaCl ₂ · 2H ₂ O is used, the pH is preferably adjusted to the 6.5 ~ 8.0 range (particularly 6.8). As another embodiment of the medium, KNO _3, Na ₂ HPO _4, MgSO ₄ · 7H ₂ O, CaCl ₂ · 2H ₂ O, Fe (III) -citrate.H ₂ O, CoCl ₂ · 6H ₂ O, CuSO ₄ 5H ₂ O, Cr ₂ O ₃ , MnCl ₂ · 4H ₂ 0, Na ₂ MoO ₄ · 2H ₂ O, SeO ₂ , biotin, thiamine, vitamine B _12, etc. can be used together.

Next, the inside of the incubator is preferably injected into the incubator while maintaining an air bubble of carbon dioxide at about 300 ~ 400 ppm. At this time, while maintaining the light 15 ~ 25W white fluorescent lamp (about 50 ± 7μEm ^-2 S ^-1 ), incubate the strain while the temperature is maintained at 20 ~ 30 ℃, especially 25 ℃, then sodium acetate (Sodium acetate) and ferrous sulfate (FeSO ₄ ㆍ 7H ₂ O) were added and the light was adjusted to a brightness of 250 ~ 350 Em ^-2 S ^-1 , and the temperature was 15 ~ 25 ℃ (especially 20 ℃) for 4 days. When cultured, hematococcus strains can be obtained from green to red gradually. Cells containing astaxanthin were cultured until their average diameter was 20-80 μm and the cell density was 10 ^{6-10 7} / mL, and the cells were suspended in water and microwaved. Astaxanthin, a natural pigment, can be obtained by extracting the solvent using ethanol and concentrating under reduced pressure with a vacuum depressurizer.

The skin whitening agent of the present invention can fully exhibit its efficacy when it contains an astaxanthin component. Specific examples of the skin whitening agent include cosmetics for skin whitening, drugs, and health functional foods.

As a first aspect of the present invention, the present invention provides a cosmetic composition for skin whitening containing astaxanthin as an active ingredient. When preparing cosmetics using astaxanthin, in addition to astaxanthin as a whitening ingredient, conventionally used ingredients such as antioxidants, stabilizers, solubilizers, vitamins, pigments, perfumes, etc. Adjuvant and carrier components are used together.

Although there is no particular limitation in the formulation of cosmetics, it is formulated with solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations and sprays. It can be used, and those skilled in the art will be able to easily adopt a suitable carrier according to the type of formulation.

In the case of the cosmetic composition, the content of astaxanthin is preferably 0.0001 to 10% by weight of the total dry weight of the cosmetic composition. When the content of astaxanthin is less than 0.0001% by weight, the skin whitening effect is weak and 10% by weight. If exceeded, the accelerated effect of the inhibitory activity of tyrosinase due to the addition of astaxanthin is not seen, and problems with dissolution may occur.

According to another aspect of the present invention, the present invention provides a dietary supplement for skin whitening comprising astaxanthin.

Here, 'health functional food' is a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc., using raw materials or ingredients having useful functions for human body, as defined in the Act on Health Functional Foods. (Refer to Article 3, Paragraph 1 of the same Act). 'Functional' refers to obtaining useful effects on health use, such as regulating nutrients or physiological effects on the structure and function of the human body. In other words, the health functional food means that it can be usefully used for health use of healthy people or semi-healthy people, not for the treatment of patients' diseases.

The skin whitening effect can be obtained just by ingesting and absorbing the health functional food containing 'astaxanthin' of the present invention, but as a functional food having a formulation such as tablets, dragees, capsules, and drinks for convenience of taking It is preferable to use.

In addition, the health functional food of the present invention may be prepared in the form of a general food, including astaxanthin ingredients, food additives, etc., which are acceptable in food, for example, beverages, medicine, kimchi, yogurt, milk, ice cream It may also be prepared in the form of a bread, rice cake and noodles. Here, the 'food additive' refers to an additive used in the production, processing or preservation of the food by the method of adding, mixing, infiltration, etc. in the food.

According to another aspect of the present invention, the present invention further provides a pharmaceutical composition comprising astaxanthin and a pharmaceutically acceptable carrier and inhibiting the activity of tyrosinase.

Suitable formulations of the pharmaceutical composition include, but are not limited to, tablets, dragees, hard or soft capsules, solutions, suspensions, emulsions, injections, suppositories, and the like. The type of carrier may be easily selected by those skilled in the art according to the formulation of the medicament, and may include one or more ingredients that can act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, disintegrating agents, and the like.

The dosage of the agent for inhibiting tyrosinase activity containing astaxanthin may vary depending on the needs of the patient, the extent of the condition to be treated, and the type of the compound to be used. . Usually, it is preferable to administer astaxanthin 0.001 to 0.10 g as a dry powder per Kg of body weight.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the present invention is not limited to the following examples, it is apparent that it can be used in the form of equivalents with various modifications.

Example 1

Culture of Hematococcus and Extraction of Natural Astaxanthin

As a component of the medium for the cultivation of Haematococcus species, sodium acetate 14.6mM, L-asparagine 2.7mM, yeast powder 2.0g / L, chloride 0.985 mM of magnesium (MgCl ₂ · 6H ₂ O), 0.036 mM of ferrous sulfate (FeSO ₄ · 7H ₂ O), and 0.135 mM of calcium chloride (CaCl ₂ · 2H ₂ O) are used. At this time, the pH is preferably adjusted to 6.8. The inside of the incubator is maintained with 350 ppm of carbon dioxide air bubbles, and the light is maintained at 25 ° C. while maintaining a brightness of 50 ± 7 μEm ⁻² S ⁻¹ with a 20 W white fluorescent lamp. Next, sodium acetate 45mM, ferrous sulfate (FeSO ₄ ㆍ 7H ₂ O) 0.45mM is added and the light is adjusted to 300 Em ^-2 S ^-1 brightness and incubated for 4 days, the temperature is 20 ℃ When maintained and cultured, hematological strains of green to red gradually become red. The average diameter of the cells containing astaxanthin is about 60 μm, and the cell density is 10 ⁷ / mL, followed by incubation in the water, microwave treatment, and solvent extraction using ethanol and vacuum. Astaxanthin which is a natural pigment was obtained by concentrating under reduced pressure with a vacuum concentrator.

Preparation of flexible lotion (skin lotion) for skin whitening

Using astaxanthin prepared by the above method, the flexible longevity was prepared by a conventional method. The components of the flexible lotion and the amount thereof are as follows.

Table 1

ingredient Unit weight% Astaxanthin One glycerin 5 1,3-butylene glycol 3 ethanol 10 Sodium hyaluronate 5 Fiji 1500 One Allantoin 0.1 Benzophenone-9 0.04 Octicdodeceth-16 0.2 Polysorbate 0.2 Distilled water Remaining amount Sum 100

Example 2

Preparation of nutritional lotion for skin whitening

Using astaxanthin prepared by the method of Example 1, the flexible cosmetic water was prepared by a conventional method. The components of the flexible lotion and the amount thereof are as follows.

[Table 2]

ingredient Content (% by weight) Astaxanthin One glycerin 5 1,3-butylene glycol 3 Sodium hyaluronate 5 Squalane 5 Glyceryl Stearate 1.5 Stearyl alcohol 1.5 Polysorbate 60 1.5 lanolin One Sorbitan stearate 0.7 Trioctanoine 1.5 Dimethicone One incense 0.01 Distilled water Remaining amount Sum 100

Example 3

Preparation of functional food (tablet) for skin whitening

After mixing with astaxanthin 5mg, lactose BP 150mg, starch BP 30mg and pregelatinized corn starch BP 15mg prepared by the method of Example 1, purified water was added appropriately and granulated into a powder. The granules were dried, mixed with 1 mg of magnesium stearate, and compressed to obtain tablets.

Example 4

Preparation of functional food (beverage) for skin whitening

Astaxanthin 2mg, food coloring 5mg, orange essence 5mg, fructose 700mg, citric acid 10mg, vitamin 5mg prepared by the method of Example 1 to prepare a composition containing a functional beverage base and then added purified water Prepared.

Example 5

Production of health functional food (syrup)

Sodium sugar (637.5 g) was dissolved in purified water (500 mL), sodium carboxymethylcellulose (2.0 g) was separately dissolved in 400 mL of purified water in a separate container, mixed with the solution of the above dissolved sugar, and methyl paraben (0.28). g) and propylparaben (0.12 g) were added to dissolve and ethanol (20 mL) was added, and the purified water was made to have a total solution volume of 1000 mL. The astaxanthin of Example 1 sieved here was suspended to obtain a syrup.

Example 6

Preparation of Ointment

Astaxanthin 5g prepared by the method of Example 1, cetyl palmitate 20g, cetanol 40g, stearyl alcohol 40g, myristan isopropyl 80g, monostearic acid sorbitan 20g, polysorbate 60g, paraoxybenzoic acid propyl 1g , 1 g of methyl paraoxybenzoate and purified water were added appropriately to prepare an ointment.

Experimental Example 1. Inhibitory effect of tyrosinase activity

In a 96 well tissue culture plate (Corning, USA), 220 μl of phosphate buffer (Sigma-Aldrich, USA) at 0.1 M pH 6.5 per well and the sample solution of kojic acid, the natural astaxanthin of Example 1 20 μl of synthetic astaxanthin and 40 μl of 1.5 mM L-tyrosine (Acros organics, USA) were added thereto. 20 μl of a tyrosinase (Sigma-Aldrich, USA) solution derived from 1,000 U / mL mushroom was added and reacted at 37 ° C. for 10 minutes. The absorbance was measured at 490 nm using an ELISA reader (Bio-Tek, USA). In the experiment of this experiment, 0.1 M phosphate buffer (pH 6.5) was added as a blank sample solution instead of the sample solution.

The above kojic acid and astaxanthin sample solution is 100, 10, 1, 0.1μM for kojic acid and synthetic astaxanthin (Sigma-Aldrich, USA), and 10, 1, 0.1μM for natural astaxanthin. Was prepared and tested.

In this experiment, the equation for determining the activity inhibition rate of tyrosinase is as follows.

Equation 1: tyrosinase inhibition rate (%) = 100-(b-b ') / (a-a') X 100

a: absorbance after reaction of blank sample liquid

b: absorbance after the reaction of the sample liquid

a ', b': absorbance measured by replacing buffer with tyrosinase in each sample

The results are shown in Table 3 below and FIG. 1.

[Table 3. Effect of Astaxanthin on Tyrosinase Inhibition]

          Active inhibition rate (%) Sample concentration (μM) Kojic acid Synthetic Astaxanthin Natural Astaxanthin (Example 1) 100 84.4 ± 8.1 - - 50 60.2 ± 4.9 30.5 ± 5.2 ^* 13.9 ± 2.7 ^** 10 50.8 ± 9.6 23.4 ± 3.8 ^* 27.5 ± 3.5 ^* 5 23.5 ± 5.8 15.1 ± 2.4 29.4 ± 2.4 2.5 13.2 ± 4.6 13.4 ± 5.1 22.2 ± 3.8 One 14.4 ± 4.5 10.2 ± 3.4 18.4 ± 3.1 0.1 9.7 ± 3.7 10.8 ± 3.7 16.5 ± 2.2 ^*

1) Mean ± S.D.

2) The asterisk means that the activity inhibition rate is significantly different within the significance level of 0.05 (*) and 0.01 (**).

As a result of the experiment, astaxanthin showed a significantly lower rate of tyrosinase activity inhibition than kojic acid at high concentrations (50, 10μM), but at low concentrations, synthetic astaxanthin did not show a difference from kojic acid, and natural astaxanthin. Rather, it showed a higher activity inhibition rate. In addition, natural astaxanthin showed higher activity inhibition than synthetic astaxanthin. Tyrosinase is responsible for oxidizing tyrosine, a type of amino acid, to produce melanin. Therefore, as confirmed in Experiment 1, it was confirmed that astaxanthin can play a large role in fulfilling the function of skin whitening by inhibiting the activity of tyrosinase.

Experimental Example 2: Experiment to inhibit melanin production using B 16 melanoma F 10 cell

B 16 melanoma F 10 cells were cultured in medium essential medium eagle (MEM) medium (Sigma-Aldrich, USA) supplemented with 10% fetal bovine serum (FBS) and trypsin-EDTA. (Gibco, USA) The cells attached to the solution were removed. After washing twice with phosphate buffered saline (PBS, phosphate buffered solution), 10% FBS + 90% MEM medium was added to prepare a cell suspension of 10 ⁴ cells / ml. Cells were attached by adding 1 ml of the above suspension per well to 24 well tissue culture plates (Corning, USA) and leaving it for 14 hours. After confirming the adhesion of the cells, the medium was removed, and the medium was added with the drug to each well (kojic acid; 100, 10, 1, 0.1 μM, the natural astaxanthin of Example 1; 10, 1, 0.1, 0.01 μM ) Was added 2 ml per well and incubated for 72 hours at 37 ℃, 5% CO ₂ air mixing incubator (Vision, Korea). After 72 hours, to remove the medium and distribute the melanin produced in the cells evenly in the solution, 2 ml of sodium hydroxide solution was added to each well and left at 99 ° C for 30 minutes to destroy the cell wall and mix evenly. The solution in the wells was measured for absorbance at 475 nm. The amount of melanin was calculated from the standard calibration curve obtained using synthetic melanin (SIGMA-ALDRICH Co., St. Louis, MO, USA) and calculated as melanin per unit cell. The results are shown in Table 4 and FIG. 2 below.

Table 4. Effect of Astaxanthin on Melanin Inhibition

Melanin (pg / cell) sample concentration (μ M ) Kojic acid Natural Astaxanthin (Example 1) 0 (untreated control) 0.602 ± 0.107 0.01 0.571 ± 0.084 0.1 0.592 ± 0.147 0.559 ± 0.083 One 0.560 ± 0.127 0.526 ± 0.070 10 0.534 ± 0.080 0.508 ± 0.073 100 0.390 ± 0.128 ^*

 1) Mean ± S.D.

 2) The asterisk means that the activity inhibition rate is significantly different within the significance level of 0.05 (*).

As a result, the melanin amount showed a tendency to decrease both the kojic acid and the natural astaxanthin when compared to the control group that did not receive the sample, and showed a significant decrease in the concentration of 100 μM kojic acid. Pigmentation on the skin, such as blemishes and freckles, is due to an ideal increase in the melanin pigment in the epidermis. Although no significant decrease was observed in the natural astaxanthin of Example 1, the concentration was decreasing to a lower value than that of kojic acid, so the experiment could not be performed due to solubility in sample preparation at a concentration of 100 μM. As a suspension, there is no problem of solubility, so a similar effect to kojic acid or a higher whitening effect is expected. In addition, astaxanthin, which is approved as a food because of the toxicity of kojic acid, is useful as a substitute for kojic acid.

The skin whitening agent of the present invention exhibits a skin whitening effect by inhibiting the activity of tyrosinase and inhibiting the production of melanin in melanocytes. Therefore, it can be usefully used as a raw material of cosmetics, health functional foods and medicines related to skin whitening or inhibition of tyrosinase activity.

Figure 1 shows the inhibition rate of tyrosinase activity of natural astaxanthin, synthetic astaxanthin and kojic acid of Example 1.

Figure 2 shows the amount of melanin per unit cell of natural astaxanthin and synthetic astaxanthin of Example 1.

Claims (7)

A skin whitening agent containing astaxanthin of Formula 1 as an active ingredient and inhibiting tyrosinase activity. [Formula 1] The natural astaxanthin according to claim 1, wherein the astaxanthin is microwave-treated by suspending hematococcus grown in the incubator under the supply of carbon dioxide and a light source in water, extracting alcohol with a solvent, and then concentrating under reduced pressure. Skin whitening agent characterized in that. The medium component for culturing hematococcus is sodium acetate, L-asparagine, yeast powder, MgCl ₂ .6H ₂ O, A skin whitening agent characterized in that it is FeSO ₄ 7H ₂ O and CaCl ₂ 2H ₂ O. A cosmetic composition for skin whitening containing astaxanthin as an active ingredient. The cosmetic composition for skin whitening according to claim 4, wherein the content of astaxanthin is 0.0001 to 10% by weight of the total dry weight of the cosmetic composition. A health functional food for skin whitening containing astaxanthin and a food acceptable food additive. A pharmaceutical composition for inhibiting tyrosinase activity comprising astaxanthin and a pharmaceutically acceptable carrier.