KR20170007518A - Composition containging amentoflavone for preventing uv-induced skin cell damage - Google Patents

Abstract

Provided is a composition for anti-aging or preventing cell damage by ultraviolet (UV) light, containing amentoflavone as an active ingredient. The composition of the present invention regulates expression of lamin A protein denatured by UV light as well as expression of phosphorylated H2AX protein. Additionally, the composition also prevents damage on skin cells and nuclear membranes of cells, and thus can be applied to cosmetic compositions or pharmaceutical compositions.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for preventing damage to skin cells caused by ultraviolet rays containing a mentoflavone,

The present invention relates to a composition for preventing skin cell damage caused by ultraviolet rays by containing amentoflavone as an active ingredient.

The lamin protein is a protein that constitutes a fibrillar network in the cell nucleus. Lamin is classified into type A and type B depending on the structure of the protein and the pattern of gene expression. Lamin A is a protein that forms a lamina immediately inside the cell nuclear membrane. There are diseases called laminopathy in the mutation of the lamin A gene, such as Hutchins Gilford progeria syndrome (HGPS), MAD (Atypical Werner's syndrome and mandibuloacral dysplasia) are known. Most progeny are those in which the cytosine at position 1824 is replaced with thymine, which produces a truncated lamin A when the lamin gene is expressed as a protein.

Typical features of progeria include sclerodermatous skin, hair loss, bone formation abnormalities, slow tooth formation, decreased growth, and loss of subcutaneous fat. In patients with progeria, abnormalities in cell nuclear structure and function have been reported, a typical example being nuclear aberration.

In addition, in recent studies, deformation of the cell nuclear structure has also been found in normal older people without progeria. Cellular nuclear abnormalities in 81-96 year olds are very similar to those of patients with progeria.

In addition, an increase in the phosphorylated H2AX protein, a marker of DNA damage, was found in the elderly, similar to that of the patient with progeria.

Therefore, it was necessary to search for a substance that regulates the modification of the cell nucleus structure, the expression of the modified lamin A protein, and the expression of the phosphorylated H2AX protein for aging inhibition research.

In order to solve the above problems, it is an object of the present invention to provide a composition for preventing cell damage or deterioration of aging caused by ultraviolet rays containing a mentoflavone as an active ingredient.

In order to accomplish the above object, the present invention provides an agent for inhibiting cell nuclear membrane damages for preventing damage to skin cells, which contains amentoflavone as an active ingredient, in an agent for preventing cellular nuclear membrane damage induced by ultraviolet irradiation.

In addition, the present invention provides a modified lamin A protein expression regulator for inhibiting skin cell damage, which contains a mentoflavone as an active ingredient, in a modified lamin A protein expression regulator induced by ultraviolet irradiation.

In addition, the present invention relates to a phosphorylated H2A histone family induced by ultraviolet irradiation, member X (H2A histone family, member X: H2AX) protein expression regulator, which comprises a phosphorylated H2AX protein expression regulator.

The present invention also provides a composition for inhibiting aging which contains amentoflavone as an active ingredient and prevents skin cell damage caused by ultraviolet rays.

The present invention also provides a cosmetic composition for preventing damage to skin cells caused by ultraviolet rays containing amentoflavone as an active ingredient.

The present invention also provides a pharmaceutical composition for preventing damage to skin cells caused by ultraviolet rays containing amentoflavone as an active ingredient.

The composition for inhibiting senescence of the present invention has an effect of controlling the expression of lamin A protein modified by ultraviolet light, controlling the expression of phosphorylated H2AX protein, preventing damage to cell nuclear membrane, and preventing skin cell damage. Thus, it can be used as a cosmetic composition or a pharmaceutical composition.

Figure 1 shows cell nuclear damage by UVB stimulation in 19 and 39 year old cells through immunofluorescence staining.
Figure 2 shows the western blot of the lamin A mutant form of progerin and phosphorylated H2AX protein in nuclear extracts of 39-year-old cells.
FIG. 3 shows the degree of nuclear damage and the degree of phosphorylation of H2AX expressed by the Lamin A defects in the UVB-treated group and the amentoflavone-treated group of 39-year-old cells.

Hereinafter, the present invention will be described in detail.

Amentoflavone is represented by the following formula (1).

 The present inventors have found that when ultraviolet rays are treated with fibroblasts, the expression of modified lamin A protein is increased and the cell nuclear membrane structure is modified. In addition, the expression of phosphorylated H2AX (H2A histone family, member X, NCBI ref. Seq. NP_002096.1) protein was increased. However, it was confirmed that not only the expression of the modified lamin A protein by the amentoflavone treatment was reduced, but also the cell nuclear membrane structure was maintained. DNA damage marker, phosphorylated H2AX protein, was also decreased by amentoflavone treatment.

The ultraviolet ray may be ultraviolet ray A, B or C.

One embodiment of the present invention provides a composition for inhibiting aging which contains a mentoflavone as an active ingredient to prevent damage to skin cells caused by ultraviolet rays, and the composition for inhibiting aging may be a cosmetic composition or a pharmaceutical composition.

An embodiment of the present invention provides a cosmetic composition or a pharmaceutical composition for preventing damage to skin cells caused by ultraviolet rays containing a mentoflavone as an active ingredient.

The pharmaceutical composition may further comprise a suitable pharmaceutically acceptable carrier, excipient or diluent.

The pharmaceutical composition of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method have. Examples of the carrier, excipient or diluent that can be contained in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. 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 form preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, (sucrose), lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the intended method, and the dose may be determined depending on the condition of the patient, The dosage, the excretion rate, the severity of the disease, the drug form, the administration time, the administration method, the administration route, and the administration period. The daily dose is 100 mg / kg to 300 mg / kg, preferably 100 mg / kg to 200 mg / kg, when the extract, fraction or compound according to the present invention is lyophilized, It may be administered once or several times a day.

Hereinafter, the present invention will be described more specifically with reference to the following examples. However, the following examples are provided for illustrative purposes only in order to facilitate understanding of the present invention, and the scope and scope of the present invention are not limited thereto.

< Example  1> Immunofluorescent staining in the 20s and 40s UVB  By stimulation Lamin  A protein expression development test

19 and 39 cells were dispensed into a 4 well chamber slide at 30,000 cells / well in a medium supplemented with 1% LSGS and 100 IU of penicillin G in 106 medium.

The next day, the medium was changed to 106 medium containing 1% fetal bovine serum (FBS). Twenty-four hours later, the wells were washed with PBS. Then, the wells were divided into two test groups. UVB treatment and UVB treatment were performed at 200 mJ / cm ² . Then, 106 medium containing 1% FBS was treated and cultured at 37 ° C for 6 days. Immunofluorescence (IF) was performed on the treated cells. Cells were washed with PBS containing 1 mM CaCl ₂ and 1 mM MgCl ₂ in each well. Cells were fixed with 3.5% paraformaldehyde at room temperature for 10 min. The immobilized cells were washed three times with PBS for 10 minutes, treated with 0.1% TritonX-100 for 5 minutes, and then washed three times with PBS for 10 minutes. Lamin A antibody (santa cruz) was diluted in PBS (PBST) containing 0.05% tween 20 at 1: 300 and reacted with O / N at 4 ° C. The cells were washed three times with PBST for 10 minutes and then reacted with a secondary antibody with FITC at 1: 400 for 1 hour at room temperature. After that, it was washed three times with PBST for 10 minutes, then mounting solution was added and cover glass was covered. The dyed slides were photographed using confocal microscopy (Ziess). The photographed results are shown in Fig. Cell nuclear damage was greater in 39-year-old cells than in 19-year-old cells.

< Example  2> In 20s and 40s cells UVB  Mutation by stimulation Lamin  A protein expression and Amentoflavone  Protective effect experiment by

106 and 100 IU of penicillin G were added to a medium of 100 g / 100 g of cells / dish for Western blotting. For immunofluorescence staining, a 4-well chamber slide 30,000 cells / well. On the next day, each cell was divided into three groups: a negative control group without UVB irradiation, a group treated with UVB only, and a group treated with UVB and amentoflavone. The cells were changed to 106-well plates containing 1% fetal bovine serum (FBS) One group was treated with amentoflavone to 5 uM and pretreated before UVB irradiation. After 24 hours, PBS was used to wash wells and dishes, and UVB was treated at 100 mJ / cm ² for each test group. Then, 106 medium containing 1% FBS was treated and cultured at 37 ° C for 6 days. The treated cells were used for immunofluorescence staining with Western blot, respectively. Western blots were prepared by lysis of cells using RIPA buffer (Sigma) and quantification of proteins using the Lowry method. Each protein was run on 4-12% Nupage gel (Invitrogen) using 10 ug of protein and transferred to NC membrane. Membrane was blocked with 5% BSA, and then the progerin (abcam) antibody and the p-H2AX (milliprore) antibody, mutants of lamin A, were diluted in 5% BSA at 1: 300 and 1: / N at 4 &lt; [deg.] &Gt; C. On the following day, each membrane was washed three times for 10 minutes with TBS (TBST) containing 0.05% tween 20, diluted with a 1: 1000 secondary antibody of HRP-conjugated mouse and rabbit, and incubated for 1 hour at room temperature . After washing three times for 10 minutes with TBST, the band was detected using ECL and LAS3000.

The results are shown in Fig. The amount of progerin (a mutant form of lamin A protein) was increased by UVB, but decreased by treatment with anthotoblast. Phosphorylated H2AX also showed the same result. Tubulin Protein loading control.

For immunofluorescence staining, cells were washed with PBS containing 1 mM CaCl2 and 1 mM MgCl2. Cells were fixed with 3.5% paraformaldehyde for 10 minutes at room temperature, and fixed cells were washed three times with PBS for 10 minutes. 0.1% TritonX-100 was treated for 5 minutes and then washed three times with PBS for 10 minutes. The primary antibody was reacted with O / N at 4 ° C by diluting 1: 300, p-H2AX antibody (millipore) 1: 200 with 0.05% tween 20 in PBS (PBST) gave. After washing three times with PBST for 10 minutes, the cells were reacted with FITC and Rhodamine-conjugated secondary antibodies at 1: 400 for 1 hour at room temperature, and then washed three times for 10 minutes using PBST. To stain the nuclei, it was stained with Propidium ipdide (PI) for about 3 minutes. After washing with PBST three times, mounting solution was added, and cover glass was covered. The dyed slides were photographed using confocal microscopy (Ziess).

FIG. 3 shows the appearance of increased nuclear damage and phosphorylated H2AX expression by treatment with UVB on 39-year-old cells after treatment with lamin A defects. In the case of amentoflavone treatment, cell nucleus shape was maintained and phosphorylated H2AX expression was also suppressed .

Formulation examples of the cosmetic composition or pharmaceutical composition according to the present invention will be described below. However, the present invention can be applied to various formulations other than the following examples.

[Formulation Example 1] Flexible lotion (skin lotion)

Flexible lotion was prepared by a conventional method according to the composition shown in Table 1 below.

ingredient Content (% by weight) Amentoflavone 2.0 glycerin 3.5 Oleyl alcohol 1.5 ethanol 5.5 Polysorbate 80 3.2 Carboxyl vinyl polymer 1.0 Butylene glycol 2.0 Propylene glycol 2.0 Preservative, fragrance Suitable amount Purified water Balance system 100

[Formulation Example 2] Nutritional lotion (Milk lotion)

Nutrition lotion was prepared according to a conventional method according to the composition shown in Table 2 below.

ingredient Content (% by weight) Amentoflavone 2.0 glycerin 3.0 Butylene glycol 3.0 Propylene glycol 3.0 Carboxyvinyl polymer 0.1 Wax 4.0 Polysorbate 60 1.5 Caprylic / capriciculiglyceride 5.0 Squalane 5.0 Sorbitacecequioleate 1.5 Cetearyl alcohol 1.0 Triethanolamine 0.2 Preservative, fragrance Suitable amount Purified water Balance system 100

[Formulation Example 3] Nourishing cream

Nutrition creams were prepared according to the compositions shown in Table 3 below in a conventional manner.

ingredient Content (% by weight) Amentoflavone 2.0 glycerin 3.5 Butylene glycol 3.0 Liquid paraffin 7.0 Beta Glucan 7.0 Carbomer 0.1 Caprylic / capriciculiglyceride 3.0 Squalane 5.0 Cetearyl glucoside 1.5 Sorbitan stearate 0.4 Polysorbate 60 1.2 Triethanolamine 0.1 Preservative, fragrance Suitable amount Purified water Balance system 100

[Formulation Example 4] Massage cream

Massage cream was prepared according to a conventional method according to the composition shown in Table 4 below.

ingredient Content (% by weight) Amentoflavone 2.0 glycerin 8.0 Butylene glycol 3.0 Liquid paraffin 45.0 Beta Glucan 7.0 Carbomer 0.1 Caprylic / capric triglyceride 3.0 Wax 4.0 Cetearyl glucoside 1.5 Sesquioleic acid sorbitan 0.9 paraffin 1.5 Preservative, coloring, fragrance Suitable amount Purified water Balance system 100

[Formulation Example 5] Pack

Nutritive creams were prepared according to the compositions listed in Table 5 below in a conventional manner.

ingredient Content (% by weight) Amentoflavone 2.0 glycerin 4.0 Polyvinyl alcohol 15.0 Hyaluronic acid extract 5.0 Beta Glucan 7.0 Allantoin 0.1 Nonyl phenyl ether 0.4 Polysorbate 60 1.2 Ethanol preservative Suitable amount Preservative, fragrance Suitable amount Purified water Balance system 100

[Formulation Example 6] Ointment in external preparation for skin

Ointments were prepared in a conventional manner according to the compositions shown in Table 6 below.

ingredient Content (% by weight) Amentoflavone 2.0 Beta-1,3-glucan 10.0 Wax 10.0 Polysorbate 5.0 Piggy 60 hardened castor oil 2.0 Sorbitan sesquioleate 0.5 vaseline 5.0 Liquid paraffin 10.0 Squalane 5.0 Sheer butter 3.0 Caprylic / capric triglyceride 5.0 glycerin 10.0 Propylene glycol 10.2 Triethanolamine 0.2 Preservative, coloring, fragrance Suitable amount Purified water Balance system 100

[Formulation Example 7] Preparation of drug (patch) for topical administration

(Patch) for topical administration was prepared according to a conventional method according to the composition shown in Table 7 below.

ingredient Content (% by weight) Amentoflavone 2.0 Beta-1,3-glucan 3.0 Diethylamine 0.7 Sodium sulphate 0.1 Polyoxyethylene lauryl ether (E.O. = 9) 1.0 Polyhydroxyethylene cetyl stearyl ether (Cetomacrogol 1000) 1.0 Viscous paraffin oil 2.5 Caprylic acid ester / capric acid ester (Cetiol LC) 2.5 Polyethylene glycol 400 3.0 Polyacrylic acid (Carbopol 934P) 1.0 Purified water Balance system 100

[Formulation Example 8] Preparation of powders

 Amentoflavone 2g

 Lactose 1g

The above components were mixed and filled in airtight bags to prepare powders.

[Formulation Example 9] Preparation of tablets

A mentoflavone 100 mg

Corn starch 100 mg

Lactose 100mg

Magnesium stearate 2 mg

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

[Formulation Example 10] Preparation of capsules

A mentoflavone 100 mg

Corn starch 100 mg

100mg of milk

Magnesium stearate 2 mg

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

[Formulation Example 11] Production of pills

A mentoflavone 1 g

Lactose 1.5g

Glycerin 1 g

Xylitol 0.5 g

After the above components were mixed, they were prepared to be 4 g per one ring by a conventional method.

[Formulation Example 12] Preparation of granules

Amento flavone 150g

Soybean extract 50mg

200 mg of glucose

Starch 600mg

After mixing the above components, 100 mg of 30% ethanol was added and the mixture was dried at 60 ° C to form granules, which were filled in a capsule.

[Formulation Example 13] Preparation of injection solution

A mentoflavone 10 mg

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ .12H ₂ O 26 mg

(2 ml) per 1 ampoule in accordance with the usual injection method.

[Formulation Example 14] Preparation of liquid agent

A mentoflavone 20 mg

10g per isomerization

Mannitol 5 g

Purified water quantity

Each component was added to purified water in accordance with the conventional liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed and then purified water was added thereto to adjust the whole volume to 100 ml. The resulting solution was filled in a brown bottle and sterilized to prepare a liquid preparation do.

Those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (6)

A composition for preventing damage to cell nuclear membrane,
The composition is intended to prevent damage to the cell nuclear membrane induced by irradiated ultraviolet rays in the skin with the modified lamin A protein,
The composition contains amentoflavone as an active ingredient,
The content of the abovementioned amentoflavone is 2% by weight, 5 [mu] M or the value between the above two values,
Wherein the composition is a cosmetic composition. In the modified lamin A (lamin A) protein expression inhibiting composition,
The composition is intended to inhibit the modified lamin A protein expression induced by ultraviolet irradiation in an increased skin of the modified lamin A protein,
The composition contains a mentoflavone as an active ingredient,
The content of the abovementioned amentoflavone is 2% by weight, 5 [mu] M or the value between the above two values,
Wherein said composition is a cosmetic composition. Phosphorylated H2A histone family, member X (H2A histone family, member X: H2AX)
The composition is intended to inhibit the expression of phosphorylated H2AX protein induced by ultraviolet irradiation in the skin with modified lamin A protein,
The composition contains a mentoflavone as an active ingredient,
The content of the abovementioned amentoflavone is 2% by weight, 5 [mu] M or the value between the above two values,
Wherein the composition is a cosmetic composition, wherein the composition is for inhibiting phosphorylated H2AX protein expression. A cosmetic composition for inhibiting skin aging caused by ultraviolet rays in skin, which contains a modified lamin A protein, which contains a mentoflavone as an active ingredient,
Wherein the content of the amentoflavone is 2% by weight, 5 [mu] M or the value of the above two values with respect to the whole composition. CLAIMS 1. A cosmetic composition for preventing damage to skin cells caused by ultraviolet rays in skin, which has increased modified lamin A protein, which contains amentoflavone as an active ingredient,
Wherein the content of the amentoflavone is 2% by weight, 5 [mu] M or the value of the above two values with respect to the whole composition. A pharmaceutical composition for preventing damage to skin cells caused by ultraviolet rays in a skin containing an aminoflavone as an active ingredient and having a modified daily dose of the amentoflavone of 100 to 300 mg / kg.

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