KR20080044612A - Composition for inhibiting c-kit protein activity containing the magnolia obovata bark extract for whitening - Google Patents

Abstract

A composition comprising an extract of Magnolia Obovata bark is provided to inhibit melanogenesis by inhibiting the activity of c-kit protein expressed in melanocyte effectively, thereby showing excellent whitening effect. A composition for inhibiting c-kit protein activity comprises 0.0001-10 wt.% of an extract of Magnolia Obovata bark as an effective ingredient, wherein the extract controls function of melanocyte by inhibiting signalling of cells in a melanocyte participated by a C-kit protein.

Description

Composition for inhibiting c-kit protein activity containing the Magnolia Obovata Bark extract for whitening}

1 is a graph of C-kit protein activity according to concentrations showing C-kit protein activity inhibition concentration-dependently by SU11248.

2 is a photograph of the induction of C-kit protein activity by SCF and inhibition of C-kit protein activity by SU11248 at the cellular level.

Figure 3 is a graph of the Japanese magnolia extract according to the present invention has an effect of inhibiting the seed kit protein activity.

Figure 4 is a photograph of the Japanese magnolia bark extract of the present invention inhibits C-kit protein activity at the cellular level.

The present invention relates to a composition for inhibiting C-kit protein activity containing Magnolia Obovata Bark Extract, and more particularly, the present invention relates to a composition of melanocytes by containing Japanese Magnolia extract as an active ingredient. By inhibiting the activity of C-kit protein, a cell surface protein, it is possible to inhibit the production of melanin (Melanin) shows an excellent whitening effect.

Recently, consumers' interest in whitening has increased due to increased ultraviolet rays due to environmental pollution or destruction of the ozone layer. However, as a large amount of side effects and hypersensitivity reactions to chemical synthetic cosmetics made of artificial compounds have been reported, attempts have been actively made to obtain cosmetic raw materials from natural materials. However, kojic acid, arbutin, or derivatives thereof, which have been identified to date, are not satisfactory for use as a cosmetic raw material due to problems in safety or stability, or the whitening efficacy when blended with the actual cosmetic raw materials. It is not. Thus, studies to find whitening active ingredients in natural products continued to find a substance that shows the inhibition of tyrosinase activity, but the problem of stability and the appropriate effective concentration remains to be solved.

The c-kit is a cell surface protein belonging to the receptor type tyrosine kinase type III family and is known as a receptor for stem cell factor (SCF). Studies in white spotting and steel mice show that SCF / c-kit functions play an important role in cell maintenance, hematopoiesis, pigmentation and reproduction. Will be in charge. As a result of the interaction between the SCF / c-kit, the C-kit protein is dimerization (dimerization), and has the activity that is phosphorylated by itself. Intracellular signal transduction then undergoes a Ras-Raf-MAP kinase cascade and finally activates Mitf (MICROPHTHALMIA-ASSOCIATED TRANSCRIPTION FACTOR) to promote tyrosinase synthesis and melanin at melanocytes. Will induce production.

SCF / c-kit signal transduction is also involved in pigmentation induced by ultraviolet B, a mechanism that likely increases SCF expression in keratinocytes and c-kit expression in melanocytes, respectively. It seems to be related to Indeed, stimulation with ultraviolet B during cell culture of keratinocytes and melanocytes increases the expression of genes and proteins in SCF and c-kit. This phenomenon was also confirmed that when treated with ultraviolet B in the epidermis of human, the exposed part was significantly increased gene and protein expression of SCF compared to the part not exposed to ultraviolet B. The same is true for Brown's Guinea pigs, an excellent model for studying melanosite function. In other words, when the inhibitory antibody against C-kit protein was injected subcutaneously, it was confirmed that pigmentation induced by ultraviolet B was completely inhibited. Therefore, it can be seen that signaling of SCF / c-kit interaction is ultimately closely related to pigmentation.

On the other hand, the Japanese magnolia bark extract used in the present invention is a state that is not known at all with respect to the inhibitory effect of melanocytes of the c-kit protein activity.

Therefore, the present inventors have studied various natural extracts to develop a substance that effectively inhibits the seed kit protein on the surface of melanocytes in the skin, so that the Japanese magnolia extract has the effect of inhibiting the activity of the seed kit protein, producing melanin. It was confirmed that the effect of suppressing the very excellent and completed the present invention.

Accordingly, it is an object of the present invention to provide a composition for inhibiting C-kit protein activity of melanocytes containing Japanese magnolia bark extract as an active ingredient.

In order to achieve the above object, the present invention provides a composition for inhibiting C-kit protein activity containing Japanese magnolia bark extract as an active ingredient.

Magnolia obovata Thunb., Which is the source of Magnolia obovata bark extract, is a deciduous tree that grows widely throughout Japan. It grows about 30m in size and large white flowers bloom in full bloom between May and June. The diameter of the flower is about 15 cm. Magnolia trees are known to be suitable for gardens and parks, but they are also widely used as building materials. Conventionally, magnolia bark has been widely used for medicinal treatments such as hemostatic astringents, gastrointestinal protective drugs, diuretics, expectorants, diarrhea, and nausea. It acts as a composition for inhibiting C-kit protein activity showing excellent whitening effect by inhibiting melanin production.

The composition for inhibiting C-kit protein activity according to the present invention contains the Japanese Magnolia bark extract in an amount of 0.0001 to 10% by weight, more preferably 0.01 to 1.0% by weight based on the total weight of the composition. This is because effective effects cannot be expected at less than 0.0001% by weight, and safety and formulation stability are difficult at more than 10% by weight.

The Japanese magnolia bark extract used in the present invention is a leaching solution of the Japanese magnolia bark, the leaching solution obtained by transfering, or a concentrate obtained by partially or totally concentrating the leaching solution, or a stagnation, whole periodic, flow extract prepared by drying the concentrate again ( Hereinafter, but is not limited to the "extract extract") and the chemical substance itself exhibiting the main effect contained in the herbal medicine.

The composition for inhibiting C-kit protein activity according to the present invention is not particularly limited in the formulation, softening longevity, astringent longevity, nourishing longevity, eye cream, nutrition cream, massage cream, cleansing cream, cleansing foam, cleansing water, Cosmetic formulations such as powders, essences and packs; Pharmaceutical formulations such as tablets, pills, capsules, suppositories, small bags, granules, powders, creams, lotions, ointments, plasters, liquid solutions, suspensions and dispersions, emulsions, syrups and the like. In addition, the composition according to the present invention can be administered orally, parenterally, rectal, vaginal, topical, transdermal, intramural, intramuscular, intraperitoneal, subcutaneous, etc., and the most preferred route of administration is transdermal administration.

On the other hand, according to the intended dosage form, the composition may be in solid, semisolid or liquid dosage form. Examples of dosage forms include, but are not limited to, tablets, pills, capsules, suppositories, small bags, granules, powders, creams, lotions, ointments, plasters, liquid solutions, suspensions and dispersions, emulsions, syrups, and the like. The active ingredient can also be encapsulated in liposomes, microparticles or microcapsules. However, the most preferred formulations are those for transdermal administration such as creams, lotions, ointments, liquid solutions, suspensions, dispersions or emulsions.

In addition, if desired, the compositions may also contain small amounts of nontoxic auxiliary substances such as wetting agents, emulsifiers, pH buffers, and the like, examples of which include but are not limited to sodium acetate, sorbitan monolaurate, triethanolamine and triethanolamine oleate. It is not limited. The compositions of the present invention may also include excipients such as stabilizers, antioxidants, binders, colorants, flavors, preservatives and thickeners.

Hereinafter, the present invention will be described in more detail with reference examples and test examples, but the present invention is not limited only to these examples.

REFERENCE EXAMPLE 1 Evaluation of C-Kit Protein Activity and Inhibition of C-Kit Protein Activity by SU11248

In order to verify the system used for activity evaluation, we observed the effect of inhibiting C-kit protein activity using SU11248 (Sugen), which binds to c-kit and inhibits activity, together with the activity of C-kit protein alone. This experiment is consistent with the method described in Panvera Z-lyte TMKinase assay kit Tyr 4 peptide, but the actual reaction temperature, time, concentration of c-kit, and concentration of ATP were optimized.

First, 2 × ATP was dispensed in 5ul portions into a 384 well dish for fluorescence measurement. Next, SU11248 made of 150nM, 50nM, 15nM, 5nM, 1.5nM, and 0.5nM, respectively, was treated using a 96-pin dish. In order to ensure the accuracy of the reaction, each sample was paired, and control experiments in the group without SU11248 / c-kit-free / phospho-peptide 0% / phosphorylated-peptide 100% Proceeded. Subsequently, 5 μl of the solution containing the c-kit and the substrate peptide thereto was dispensed, followed by reaction at 37 ° C. for 45 minutes. After the reaction was completed, the developer solution was dispensed by 5ul for each reaction at room temperature for 40 minutes, and finally, 5ul of the stop solution was dispensed to complete the experiment.

The final concentration of each material used in the reaction corresponds to 500uM ATP, 1.5ng c-Kit (Kinase), 2uM peptide, SU11248 corresponds to the final concentration of 30nM, 10nM, 3nM, 1nM, 0.3nM, 0.1nM.

On the other hand, by measuring the fluorescence value of the reaction was completed samples to derive the inhibition rate of SU11248 on the C-kit protein activity. The two fluorescence values were read using light activated through 405 nm and 535 nm filters, respectively, and the activation levels of enzymes were measured by calculating the ratio therebetween, and the results are shown in FIG. 1.

As shown in FIG. 1, it was confirmed that SU11248 inhibited the activity of c-kit in a concentration-dependent manner.

REFERENCE EXAMPLE 2 Evaluation of C-Kit Protein Activity and Confirmation of C-Kit Protein Activity Inhibitory Effect at the Cell Level by SU11248

In order to verify the system used for activity evaluation, we observed the effect of inhibiting the C-kit protein activity at the cellular level by SU11248, which binds to the c-kit and inhibits the activity, in addition to the C-kit protein alone activity. The cell line was HM3KO, a human-derived melanosite, and the culture was carried out under conditions of MEM medium, 37 ° C., and 5% CO ₂ to which 10% placental serum was added. Cultured HM3KO cells were detached with 0.25% Trypsin-EDTA and placed on the 6-well plate again with the same number (2.4 × 10 5 cells / well) and left for 24 hours. Thereafter, all of the medium was removed, and then replaced with MEM medium containing 1% fetal placenta serum, followed by incubation for another 24 hours. On the third day, SU11248 was again treated with a final concentration of 50 nM after again changing to MEM medium with 1% fetal placenta serum. Subsequently, SCF (Stem cell factor), which is attached to H-M3KO cells to c-kit specificly and induced activity for 24 hours, was added at a final concentration of 50 ng / ml for 15 minutes to induce c-kit activity. No treatment of each group / SCF-only group / SU11248 pretreatment and SCF-treated group were used to compare the increase of c-kit activity alone and the inhibitory effect by SU11248. The results are shown in FIG. 2.

From the results of FIG. 2, it was confirmed that the pretreatment with SU11248 and the treatment with SCF inhibited the activity of c-kit as compared with the treatment with SCF alone.

Experimental Example 1 Inhibitory Effect of Seed Kit Protein Activity by Japanese Magnolia Extract

From Reference Example 1, it was confirmed that C-kit protein activity measurement and activity inhibition by a specific inhibitor can be evaluated. Based on this, an experiment was performed to evaluate whether the Japanese magnolia bark extract used in the present invention could inhibit the activity of C-kit protein under the same reaction conditions as in Reference Example 1.

The reaction conditions used in the experiment was the same as in Reference Example 1, but the final concentration of the Japanese magnolia bark extract used to see the activity inhibition was equivalent to 320ppm, 160ppm and 80ppm. After the reaction was completed, the value obtained by the fluorimeter was calculated and the degree of inhibition of C-kit protein activity is shown in FIG. 3.

As can be seen in Figure 3, it was confirmed that the Japanese magnolia bark extract used in the present invention has an effect of inhibiting the activity of the seed kit protein concentration-dependently.

Test Example 2 Inhibition of C-Kit Protein Activity at the Cell Level by Japanese Magnolia Extract

From Reference Example 2, it was confirmed that C-kit protein activity measurement and inhibition of activity by a specific inhibitor can be evaluated at the cellular level. Based on this, an experiment was conducted to evaluate whether the Japanese magnolia bark extract contained in the external preparation composition for skin whitening of the present invention can inhibit the activity of C-kit protein under the same reaction conditions as in Reference Example 2. The reaction conditions were the same as in Reference Example 2, but the final concentration of the materials used in the present invention corresponded to 16 ppm, 8 ppm, and 4 ppm. The results are shown in FIG.

From the results of Figure 4, it can be seen that the Japanese magnolia bark extract used in the present invention effectively inhibits the activity of c-kit. In particular, c-kit inhibition of melanocytes inhibits tyrosinase production and consequently inhibits melanin synthesis.

<Formulation Example 1: Nutritious Longevity (Milk Skin Lotion)>

Nutrients according to the present invention was prepared in the composition of Table 1.

ingredient Content (% by weight) Purified water To 100 glycerin 8.0 Butylene glycol 4.0 Hyaluronic acid extract 5.0 Beta Glucan 7.0 Carbomer 0.1 Japanese Magnolia Extract 0.05 Caprylic Capric Triglycerides 8.0 Squalane 5.0 Cetearyl Glucoside 1.5 Sorbitan stearate 0.4 Cetearyl Alcohol 1.0 antiseptic Quantity incense Quantity Pigment Quantity Triethanolamine 0.1

<Formulation Example 2: Nutrition Cream>

Nutritional cream according to the present invention was prepared in the composition of Table 2.

ingredient Content (% by weight) Purified water To 100 glycerin 3.0 Butylene glycol 3.0 Liquid paraffin 7.0 Beta Glucan 7.0 Carbomer 0.1 Japanese Magnolia Extract 3.0 Caprylic Capric Triglycerides 3.0 Squalane 5.0 Cetearyl Glucoside 1.5 Sorbitan stearate 0.4 Polysorbate 60 1.2 antiseptic Quantity incense Quantity Pigment Quantity Triethanolamine 0.1

<Formulation example 3: massage cream>

Massage cream according to the present invention was prepared in the composition of Table 3.

ingredient Content (% by weight) Purified water To 100 glycerin 8.0 Butylene glycol 4.0 Liquid paraffin 45.0 Beta Glucan 7.0 Carbomer 0.1 Japanese Magnolia Extract 1.0 Caprylic Capric Triglycerides 3.0 Beeswax 4.0 Cetearyl Glucoside 1.5 Sesqui oleic acid sorbitan 0.9 Vaseline 3.0 antiseptic Quantity incense Quantity Pigment Quantity paraffin 1.5

<Formulation Example 4: Pack>

The pack according to the invention was prepared in the composition of Table 4 below.

ingredient Content (% by weight) Purified water To 100 glycerin 4.0 Polyvinyl alcohol 15.0 Hyaluronic acid extract 5.0 Beta Glucan 7.0 Allantoin 0.1 Japanese Magnolia Extract 0.5 Nonyl Phenyl Ether 0.4 Polysorbate 60 1.2 antiseptic Quantity incense Quantity Pigment Quantity ethanol 6.0

<Formulation Example 5: Ointment>

Ointments according to the invention were prepared in the composition of Table 5 below.

ingredient Content (% by weight) Purified water To 100 glycerin 8.0 Butylene glycol 4.0 Liquid paraffin 15.0 Beta Glucan 7.0 Carbomer 0.1 Japanese Magnolia Extract 1.0 Caprylic Capric Triglycerides 3.0 Squalane 1.0 Cetearyl Glucoside 1.5 Sorbitan stearate 0.4 Cetearyl Alcohol 1.0 antiseptic Quantity incense Quantity Pigment Quantity Beeswax 4.0

As described above, the composition for inhibiting C-kit protein activity according to the present invention contains the Japanese magnolia bark extract as an active ingredient and effectively inhibits the activity of C-kit protein expressed in melanocytes to produce melanin. Because it can inhibit the excellent whitening effect.

Claims (4)

A composition for inhibiting C-kit protein activity, comprising a Japanese Magnolia Obovata Bark Extract as an active ingredient. The composition of claim 1, wherein the active ingredient is contained in an amount of 0.0001 to 10% by weight based on the total weight of the composition. The composition of claim 1, wherein the composition has the use of skin whitening. The composition of claim 1, wherein the Japanese Magnolia bark extract regulates the function of melanocytes by inhibiting signal transduction of cells in the melanocytes involved in the seed kit protein.

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