KR100589716B1 - Composition for inhibiting melanin synthesis - Google Patents

Abstract

The present invention relates to a composition for inhibiting melanin production. In particular, the present invention inhibits the expression of microphthalmia-associated transcription factor (MITF) and tyrosinase, while simultaneously inhibiting tyrosinase activity, and exhibits a synergistic melanin production inhibitory effect when used in combination, hinokithiol, and green tea extract or EGCG (epigallocatechin gallate) It provides a composition for inhibiting melanin production comprising as an active ingredient.

Hinokithiol, green tea extract, EGCG, whitening, tyrosinase, melanin, MITF

Description

Composition for inhibiting melanin production {COMPOSITION FOR INHIBITING MELANIN SYNTHESIS}

1a and 1b are graphs and cell photographs showing the tyrosinase inhibitory effect of EGCG (epigallocatechin gallate), hinokithiol, and kojic acid.

Figure 2a is a graph showing the rate of melanin synthesis according to the combination treatment with kojic acid and EGCG.

Figure 2b is a graph showing the rate of melanin synthesis according to the combination of hinokithiol and EGCG.

Figure 3 is a photograph observing the degree of melanin synthesis in Mel-Ab cells when treated with hinokitiol in combination with EGCG or green tea extract (green tea polyphenol, GTP).

Figure 4 is a photograph confirming the expression of microphthalmia-associated transcription factor (MITF) and tyrosinase after treatment with kojic acid, EGCG and hinokithiol.

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to a composition for inhibiting melanin production, and more particularly, to a composition having a markedly elevated melanin synthesis inhibitory effect by inhibiting MITF expression and inhibiting tyrosinase expression by simultaneously treating green tea extract and EGCG with hinokithiol. It is about.

[Private Technology]

Hyperpigmentation of the skin can be caused by various factors such as hormonal abnormalities, genetic diseases, and ultraviolet irradiation after the inflammatory response of the skin, and the main factors include melanin pigment synthesis abnormalities and distribution abnormalities.

The main function of melanin is to remove oxygen radicals and protect the skin from the damage caused by it. Therefore, high levels of melanin mean that it has an effective response system to protect the skin from physical and chemical toxic substances. Melanogenesis is achieved by the action of enzymes and spontaneous oxidation after tyrosine (Tyrosine) is converted to dopaquinone by Tyrosinase in melanocytes.

Methods of suppressing melanin production can be broadly divided into the following.

First, it blocks UV rays and removes the main cause of melanin production. This method allows the cosmetic composition to contain a light scattering agent or a light blocking agent and good results can be expected.

Second, it inhibits the production of melanin by inhibiting the synthesis of core carbohydrates required for tyrosinase activity such as glucosamine.

Third, kojic acid or arbutin interferes with the function of tyrosinase, an enzyme involved in melanogenesis.

Fourth, by using a substance having a specific toxicity against melanocytes that produce melanin, such as hydroquinone to prevent cell division.

In addition, there is a method of reducing the produced melanin to decolorize.

Currently, many studies are being conducted to find an effective whitening composition, and most of the studies have focused on substances that directly inhibit tyrosinase enzymes involved in melanin synthesis. Recently, however, it has been suggested that in addition to direct inhibition of tyrosinase enzymes, whitening effects can be expected by regulating signal transduction involved in tyrosinase expression (Briganti S, Camera E, Picardo M. Pigment Cell Res , 2003, 16 ( 2): 101-10). For example, it is known that melanin production increases when c-AMP is increased, and melanin production decreases when Akt or ERK pathways are activated. Tyrosinase has been reported to be produced under the control of the transcription factor MITF (microphthalmia-associated transcription factor) when affected by external stimuli or hormones (DS Kim, ES Hwang, JE Lee, SY Kim, SB Kwon, and KC Park.J Cell Sci . 2003; 116: 1699-706). MITF is an important transcription factor for the expression of tyrosinase gene. When MITF is expressed a lot, the production of tyrosinase is increased, which eventually increases melanin production.

Therefore, the development of a substance capable of inhibiting tyrosinase production through the inhibition of MITF expression, which is a transcription factor of tyrosinase enzyme, is required to overcome the conventional concept of only inhibiting the activity of tyrosinase enzyme.

Accordingly, an object of the present invention is to provide a composition capable of inhibiting melanin production by inhibiting MITF expression and simultaneously inhibiting tyrosinase expression and activity.

It is another object of the present invention to provide a composition capable of inhibiting MITF expression.

It is another object of the present invention to provide a composition that is harmless to the human body and has an excellent whitening effect.

In order to achieve the above object, the present invention provides a composition for inhibiting melanin production, including hinokithiol, and green tea extract or EGCG (epigallocatechin gallate) as an active ingredient.

In another aspect, the present invention provides a composition for inhibiting the expression of microphthalmia-associated transcription factor (MITF) comprising a hinokithiol or EGCG (epigallocatechin gallate) as an active ingredient.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention, while searching for a substance that inhibits MITF expression, confirmed that hinokitiol and EGCG not only significantly inhibit MITF expression, but also inhibit tyrosinase expression and inhibit activity, and also use EGCG or green tea extract in combination with hinokitiol. When used, it was confirmed that the melanin synthesis is significantly reduced to complete the present invention based on this.

The present invention relates to a composition for inhibiting melanin production comprising (a) hinokithiol and (b) EGCG or green tea extract as an active ingredient.

The hinokithiol, EGCG and green tea extracts of the present invention may be prepared from natural plants by known methods, respectively, or may be used by purchasing a commercially available product. For example, green tea extract may be prepared by mixing the green tea leaf powder in 0.1 to 1 kg per liter of water, filtering and concentrating it under reduced pressure. Green tea extract contains flavonoids such as antoxanthin and anthocyanin, and catechins and leuco anthocyanins, which are polyphenols.

The composition for inhibiting melanin production of the present invention may include the active ingredient alone, but it may be contained in 0.001 to 50% by weight, preferably 0.001 to 5% by weight, and further includes other pharmacologically acceptable substances. can do. In addition, the mixing ratio of the hinokithiol and EGCG or green tea extract in the active ingredient may be 1: 0.001 to 5 weight ratio, but is not limited thereto.

The pharmacologically acceptable materials are excipients, carriers or diluents commonly used in pharmaceuticals or cosmetics, such as, but not limited to, water, saline, glycerol and the like.

Melanin production inhibition composition of the present invention can be used as a medicament, cosmetics or cleaning agents, the formulation thereof may be applied differently depending on the method and purpose of use. Examples of formulations include lotions, solutions, emulsions, emulsions, suspensions, tablets and capsules. When used as a cosmetic, it can be used in the form of softening lotion, milk lotion, nourishing cream, massage cream, essence, cleansing foam, cleansing water, pack or body oil, and basic cosmetics such as foundation, lipstick, mascara or makeup base Do. When used as a detergent, it can be used as a face wash and bath.

In addition, the present invention relates to a composition for inhibiting MITF expression comprising hinokithiol or EGCG as an active ingredient, which may include only the active ingredient or 0.001 to 5% by weight of the active ingredient and the remaining amount of pharmacologically acceptable substance. have. The composition for inhibiting MITF expression of the present invention can be used for the same use as the composition for inhibiting melanin production mentioned above.

As confirmed in one embodiment of the present invention, hinokithiol and EGCG not only inhibit MITF and tyrosinase expression (FIG. 4), but also show simultaneous effects of inhibiting tyrosinase activity (FIGS. 1A and 1B). In addition, when EGCG or green tea extract is used in combination with hinokithiol, it shows a synergistic melanin production inhibitory effect (FIGS. 2B and 3), which is much better than EGCG in combination with kojic acid (FIG. 2A). .

Accordingly, the present invention provides a whitening composition comprising green tea extract / or EGCG and hinokithiol. The whitening composition can be used as a cosmetic or pharmaceutical composition to reduce melanin deposition.

Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention, but the protection scope of the present invention is not limited to the following examples.

Example 1: Sample Preparation

1-1. Green tea extract

The green tea leaves were washed with purified water, dried and then powdered. 1 kg of green tea powder was added to 5 L of water, extracted at 20 ° C. for 5 days, filtered through a 300 mesh filter cloth, left at 10 ° C. for 10 days, and then aged at low temperature. The filtrate was concentrated under reduced pressure and dried to prepare a green tea extract.

1-2. Hinoki Thiol

Hinokithiol was purchased from Sigma.

1-3. EGCG

EGCG was purchased from Sigma.

Comparative example

Kojic acid was purchased from Sigma.

Experimental Example 1 Inhibition of Tyrosinase Activity

Melanin concentrations and tyrosinase activity inhibition rates are characterized by pigmented intermediates (dopachrome, 5,6-inhydroxyhydroxydole-2-carboxylic acid (DHICA :) that are produced by tyrosinase acting on tyrosine to produce melanin. 5,6-dihydroxyindole-2-carboxylic acid), and 5,6-dihydroxyindole (DHI: 5,6-dihydroxyindole).

Inhibition of tyrosinase activity by green tea extract, EGCG and hinokthiol, respectively, or a mixture of two species was performed by known methods (Vanni A, Gastaldi D, Giunata G et al., Annali Di Chimica , 1990; 80: 35-60). Confirmed by.

40 µl of L-tyrosine solution (manufactured by Sigma) was added to each well of a 96-well plate, followed by addition of hinokithiol (0.01-25 µg / ml), followed by 20 µl of 50 µg / ml tyrosinase (manufactured by Sigma). It was. After reacting at 37 ° C. for 30 minutes, the absorbance was measured at 490 nm, and then substituted into the following Formula 1 to calculate the tyrosinase activity inhibition rate. In addition, the L-tyrosine solution was used as a negative control, the treatment with kojic acid was used as a positive control. Each result is shown in Table 1 below.

(Calculation 1)

A: absorbance at 490 nm of control group

B: absorbance at 490 nm of experimental group

Experimental material  Tyrosinase activity inhibition rate (%) 0.01 μg / ml 0.1 μg / ml 1 μg / ml 10 μg / ml 25 μg / ml Kojic acid 63.1% 89.9% Green Tea Extract -2.1 -2.7 13.2 12.4 EGCG 1.1 -3.5 27.6 34.8 Hinoki Thiol -0.1 21.2 85.4 94.2 97.3 Hinokithiol + green tea extract (0.1 µg / ml) 2.5 22.1 Hinokithiol + Green Tea Extract (1µg / mL) 1.7 17.5 Hinokithiol + EGCG (0.1 μg / ml) -0.4 18.9 Hinokithiol + EGCG (1 μg / ml) 1.0 26.5

In Table 1, the tyrosinase activity inhibition rate was the best of the hinokithiol, followed by the high inhibition rate of kojic acid. On the other hand, green tea extract and EGCG showed little inhibition of tyrosinase activity at low concentrations. In addition, when a combination of green tea extract or EGCG in the hinokithiol, it was confirmed that there is no synergistic effect on the inhibition of tyrosinase activity.

In addition, inhibition of tyrosinase activity at the standard concentration of each sample was confirmed. The results are shown in Figures 1a and 1b. In FIG. 1A, kojic acid hardly inhibited tyrosinase activity at 100 μM concentration, but EGCG showed a slightly higher inhibitory effect than 100 μM concentration of kojic acid at 10 μM concentration. In addition, hinokithiol showed an excellent inhibitory effect on tyrosinase activity at 10 μM, which is 10 times lower than kojic acid. In the cell photograph of FIG. 1B, the same results as in FIG.

Experimental Example 2: Experiment to inhibit melanin synthesis in modified melanoma cells

end. When processing alone

For melanin synthesis inhibition experiments, Mel-Ab cells, modified melanoma cells, were seeded in a 6 well culture dish, and samples were incubated for 5 days with varying concentrations. Cultured cells were collected to quantify the amount of each protein, and then the same amount of protein was added to 1 ml of 1N sodium hydroxide solution and boiled at 100 ° C. for 30 minutes to destroy cells. The absorbance of the suspension was measured at 400 nm using an ELISA reader. In addition, Mel-Ab cells without the sample was used as a control, and the same experiment was conducted as described above. Kojic acid (Sigma Co., Ltd.), which is known to have a melanin synthesis inhibitory function, was used as a positive control sample, in order to maximize the effect of kojic acid. The same procedure was followed except that the amount of the material used in the experiment was up to 100 μM. The experimental results are shown in Table 2.

division Melanin synthesis rate (% of control) Kojic acid EGCG Hinoki Thiol 0 100.0 100.0 100.0 0.1 μM 83.9 1 μM 95.2 83.8 61.5 10 μM 84.4 73.8 53.3 100 μM 74.5

In Table 2, it was confirmed that the hinokithiol is significantly higher melanin synthesis inhibitory effect than EGCG and kojic acid.

I. When combined treatment

After combining EGCG with kojic acid or hinokithiol to Mel-Ab cells, the inhibition rate of melanin synthesis was confirmed. Experimental method was carried out in the same manner as the above.

Figure 2a is a graph showing the melanin synthesis rate according to the combination treatment with kojic acid and EGCG, EGCG treated 0, 1 and 10 μM and kojic acid treated 0 or 100 μM, respectively. As a result, the melanin synthesis rate in the co-treatment with kojic acid and EGCG was slightly lower than that in the co-treatment alone, but no synergistic effect was observed in the co-treatment with kojic acid and EGCG.

Figure 2b is a graph showing the rate of melanin synthesis according to the combination of hinokithiol and EGCG, the melanin synthesis rate in the EGCG 1 μM alone treatment group, the hinocytol 1 μM treatment group and EGCG 1 μM + hinokthiol 1 μM combination treatment group. . Experimental results showed that when EGCG was combined with the hinocytol, the melanin synthesis rate was significantly reduced compared to the treatment group. Therefore, it can be seen that the combination of hinokithiol and EGCG has a synergistic effect on melanin synthesis inhibition.

FIG. 3 is a photograph of melanin synthesis in Mel-Ab cells when hinocthiol is used in combination with EGCG or green tea extract (green tea polyphenols, GTP), and it can be seen that remarkably inhibits melanin synthesis in the combination treatment. .

Experimental Example 3: MITF expression suppression experiment

In order to confirm the melanin synthesis inhibitory mechanism of kojic acid, EGCG and hinokithiol, the inhibition rate of MITF expression according to each sample was confirmed.

Cultured Mel-Ab melanocytes were treated with 10 μM EGCG, 10 μM hinokthiol, and 100 μM kojic acid, respectively, and after 3 days, changes in the expression patterns of MITF and tyrosinase were observed.

Figure 4 is a photograph confirming the MITF and tyrosinase expression rate according to kojic acid, EGCG and hinokithiol treatment, "con" is a non-treated group, "1" is EGCG 10 μM treated group, "2" 100 μM treated kojic acid group And "3" is a hinokithiol 10 μM treatment group. In the experimental results, EGCG and hinokithiol inhibited the expression of MITF as well as tyrosinase expression at a concentration 10 times lower than kojic acid. On the other hand, it was observed that kojic acid did not inhibit MITF expression at all, and the tyrosinase expression inhibition rate was also significantly lower than that of EGCG and hinokthiol. Thus, it was found that EGCG and hinokithiol inhibit the synthesis of tyrosinase by inhibiting the expression of MITF.

Example 2 Preparation of Softener Longevity (Skin) Containing Hinokithiol and Green Tea Extract

According to the conventional method, 0.1% by weight of the hinokithiol, 1% by weight of the green tea extract, 3% by weight of glycerol, 5% by weight of ethanol, 2% by weight of propylene glycol, flavors, and the remaining amount of purified water to mix the hinokthiol and green tea extract Containing softening water containing was prepared.

Example 3: Preparation of Converging Cosmetics Containing Hinoki Thiol and Green Tea Extract

According to a conventional method, 0.5% by weight of hinokithiol, 1% by weight of the green tea extract, 4% by weight of beeswax, 4% by weight of liquid paraffin, 4% by weight of squalene, 3% by weight of glycerin, trace amount of preservative, trace amount of purified water To prepare astringent cosmetic water containing the hinokithiol and green tea extract.

Example 4: Preparation of nourishing cream containing hinokithiol and green tea extract

According to a conventional method, 0.1% by weight of hinokithiol, 0.5% by weight of the green tea extract, 6% by weight of propylene glycol, 4% by weight of glycerin, 5% by weight of liquid paraffin, 3% by weight of squalene, 1.5% by weight of polysorbate 60, and The remaining amount of purified water was mixed to prepare a nourishing cream containing hinokithiol and green tea extract.

Example 5 Preparation of Massage Cream Containing Hinokithiol and Green Tea Extract

According to the conventional method, 0.5% by weight of hinokithiol, 0.2% by weight of the green tea extract, 7% by weight of petrolatum, 10% by weight of liquid paraffin, 2% by weight of beeswax, 2.5% by weight of polysorbate, 3% by weight of squalene, glycerin 4 A massage cream containing hinokithiol and green tea extract was prepared by mixing wt%, propylene glycol 6 wt%, a preservative trace amount, and the remaining amount of purified water.

Example 6 Preparation of Essence of Hinokthiol and EGCG

According to the conventional method, 0.1% by weight of hinokthiol, 0.01% by weight of EGCG, 2% by weight of propylene glycol, 4% by weight of glycerine, 7% by weight of ethanol, a preservative amount, a flavor amount, and a residual amount of purified water were mixed to prepare the hinokthiol and EGCG. Essence containing was prepared.

Example 7 Preparation of a Pack Containing Hinokthiol and EGCG

According to a conventional method, 0.5% by weight of hinokthiol, 0.01% by weight of EGCG, 6% by weight of propylene glycol, 4% by weight of glycerin, 2% by weight of beeswax, 30% by weight of liquid paraffin, trace amounts of preservatives, flavors, and the balance of purified water Packs containing hinokithiol and EGCG were prepared.

As described above, the hinokitiol and EGCG of the present invention not only inhibits MITF and tyrosinase expression, but also has a simultaneous effect of inhibiting tyrosinase activity, and synergistic melanin when EGCG or green tea extract is used in combination with hinokitiol Inhibition of production. Therefore, the composition comprising the hinokithiol and EGCG or green tea extract of the present invention can be used as a cosmetic or medicament for reducing melanin pigmentation.

Claims (5)

Hinokithiol; And Green tea extract or epigallocatechin gallate (EGCG); As a composition for inhibiting melanin production comprising as an active ingredient, The composition for inhibiting melanin production that comprises the hinokithiol and green tea extract or EGCG 1: 0.001 to 5 by weight. delete The composition of claim 1, wherein the composition comprises 0.001 to 5% by weight of the active ingredient. A composition for inhibiting expression of a microphthalmia-associated transcription factor (MITF) comprising hinokithiol or EGCG as an active ingredient. The composition of claim 4, wherein the composition comprises 0.001 to 5% by weight of hinokithiol or EGCG.

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