KR102141095B1 - Use of Mimosa pudica extracts for manufacture of composition for inhibiting MMP2 gene expression and collagen degradation - Google Patents

Abstract

The present invention provides the use of a mimosa extract for preparing a composition that inhibits the expression of matrix metalloproteinase 2 (MMP2) gene and the use of a mimosa extract for preparing a composition that inhibits collagen degradation. The mimosa extract according to the present invention is prepared by extracting mimosa with a solvent of water, alcohols, or an alcohol-water mixture. The mimosa extract inhibits the gene expression of MMP2 in skin fibroblasts, thereby reducing the degradation of collagen in the skin, thereby improving skin elasticity and density, reducing fine lines and wrinkles of the skin, and delaying skin aging.

Description

Use of Mimosa pudica extracts for manufacture of composition for inhibiting MMP2 gene expression and collagen degradation}

The present invention relates to the use of the mimosa extract, and more particularly, to the use of the mimosa extract for preparing a composition that inhibits the expression of matrix metalloproteinase 2 (MMP2) gene and collagen degradation.

Collagen (collagen) is a very high protein content in the body of the animal, accounting for about 25% of the total protein in the body. Collagen is mainly distributed in the extracellular matrix of connective tissue such as ligaments, tendons, cartilage, and cornea in the animal body, and is the most important protein in connective tissue and accounts for about 20% to 30% of connective tissue. Collagen has a fibrous structure and can provide the necessary mechanical support and strength for connective tissue, and is a very important structural protein related to blood clotting, wound healing, and tissue repair. Collagen in the human body can be divided into various types according to differences in polypeptide composition. Among them, collagen types 1, 3, and 4 are most closely related to the skin structure. The dermal layer contains collagen types 1 and 3, which are involved in maintaining moisture and improving elasticity of the skin, and in the basement membrane below the epithelial tissue, collagen of type 4, which is used for moisture transfer and maintenance of nutrients in the skin. Related.

With the increase of age and long-term irradiation of ultraviolet rays, collagen in the skin is gradually lost and the water content of the skin decreases, and the dry state of the skin further leads to problems such as loss of elasticity of skin, wrinkle formation and pore enlargement. Therefore, supplementing collagen in the skin, increasing the synthesis of collagen, and reducing the degradation of collagen is already a research focus to delay skin aging and maintain skin health. The method of applying a non-product containing collagen directly to the skin surface has a problem in that it is unable to effectively supplement collagen in the depth of the skin due to the blocking action of the skin epidermal layer. On the other hand, when ingesting collagen by the oral administration method, the collagen of the polymer is broken down into amino acids of small molecules in the digestive system, and the above amino acids ingested in each part of the human body are not synthesized as essential collagen, so taking collagen by mouth How to do so will be limited by the effect of improving the collagen synthesis of the skin.

Accordingly, it is urgently necessary to develop a new composition capable of effectively suppressing the degradation of collagen to reduce the loss of collagen and prevent skin aging.

Accordingly, an object of the present invention is to provide a use of Mimosa pudica extract for preparing a composition that inhibits the expression of matrix metalloproteinase 2 (MMP2) gene.

In addition, another object of the present invention is to provide a use of a mimosa extract for preparing a composition that inhibits collagen degradation, wherein the mimosa extract is obtained by extracting mimosa using a solvent.

In one embodiment according to the present invention, the solvent is water, alcohols, or an alcohol-water mixture, and the liquid-solid ratio of the solvent and mimosa is 5 to 20:1 to 5, and the extraction is 50°C to It proceeds at 100°C.

In one embodiment according to the present invention, the mimosa extract is a mimosa water extract, and the concentration is at least 0.5 mg/mL.

In one embodiment according to the present invention, the collagen is type 4 collagen.

The mimosa extract obtained by extracting a mixture of water, alcohols, and alcohol-water according to the present invention can suppress the gene expression of MMP2 in skin fibroblasts, thereby reducing the degradation of type 4 collagen in the extracellular matrix and finally The effect of increasing the collagen content in the skin can be achieved, thereby improving skin elasticity and density and fine lines of the skin. And reducing wrinkles and delaying skin aging. Therefore, the mimosa extract according to the present invention can be used for the preparation of a composition that inhibits the expression of MMP2 gene and the degradation of collagen, and the composition may be in powder form, granule form, liquid form, gel form or paste form, and food It can be prepared as a beverage, drug, reagent, or nutritional supplement, and can be used on an individual by oral administration or application to the skin.

Hereinafter, an exemplary embodiment of the present invention will be described in detail with reference to the accompanying drawings. The examples described in the following description are only for explaining the features and use of the present invention, and are not intended to limit the scope of the present invention, and all persons who are familiar with the present technology do not depart from the spirit and scope of the present invention. Changes and modifications can be made. Therefore, the protection scope of the present invention should be defined by the scope of the appended claims.

Figure 1 shows the relative amount of collagen produced in human skin fibroblasts and human skin fibroblasts of the control treated by the mimosa extract according to the present invention.
Figure 2 shows the relative expression of the MMP2 gene in human skin fibroblasts treated by the mimosa extract according to the present invention.

The present invention provides the use of a mimosa extract for preparing a composition that inhibits collagen degradation. The mimosa extract according to the present invention is obtained by extracting mimosa using a solvent, wherein the solvent is water, alcohols, or an alcohol-water mixture, and the liquid-solid ratio of the solvent and the mimosa is 5-20 :1 to 5, and the extraction proceeds at 50°C to 100°C. By experiments on collagen content and analysis of the expression level of collagen degradation related genes, the mimosa extract according to the present invention can improve the collagen production of human skin fibroblasts and the increase in production is related to the decrease in the expression level of the MMP2 gene. It was proved. Therefore, the mimosa extract according to the present invention can also be used for preparing a composition that inhibits MMP2 gene expression.

Justice

Numerical values used herein are approximate and all experimental data are presented within a range of 20%, relatively preferably within a range of 10%, more preferably within a range of 5%.

Materials and methods

material

Buy Eagle's minimum essential medium (weak with MEM medium), fetal bovine serum (weak with FBS), including Earle's equilibrium salt solution from Gibco, non-essential amino acids, sodium bicarbonate, pyruvate Sodium and phosphate buffer (phosphate buffered saline, weak with PBS) were purchased.

Cell culture

The following examples were conducted using human skin fibroblast CCD-966SK (BCRC 60153). Human skin fibroblasts were cultured in MEM medium (hereinafter referred to as cell medium) to which 10% FBS, 0.1 mM non-essential amino acid, 1.5 g/L sodium bicarbonate, and 1 mM sodium pyruvate were added at 37° C. and 5% carbon dioxide. .

Collagen content experiment

In the present invention, the content of collagen secreted by cells was measured using a soluble collagen assay kit (Sircol Soluble Collagen Assay Kit, Bicolor), and the approximate procedure is as follows. First, 1000 μL of cell medium is taken, mixed with 200 μL of collagen separation/concentration reagent, and then incubated for a certain period of time with repeated inversion at 4° C., and then the mixture is centrifuged for 10 minutes at a rotation speed of 12000 rpm. The supernatant was removed. Subsequently, the remaining precipitate was mixed with 1000 μL of Sircol colorant for 30 minutes, and again, the colorant mixture was centrifuged for 10 minutes at a rotation speed of 12000 rpm to form a collagen precipitate. After removing the supernatant, the collagen precipitate is first washed with an acidic salt solution of 750 μL, the excess liquid is removed, the collagen precipitate is dissolved with 250 μL alkaline reagent, and then the collagen is analyzed using an enzyme immunoassay (ELISA reader, BioTek). The absorbance of the solution was measured at a wavelength of 555 nm. Compared to the standard curve of the water-soluble collagen standard, the collagen content in the cell medium to be measured can be calculated. Statistical analysis was determined using Student's t distribution in Excel software.

Gene expression analysis

In the present invention, the gene expression level of enzymes involved in the degradation of collagen in cells was measured through a quantitative polymerase chain reaction (which is weak as Q-PCR), and the approximate sequence is as follows. According to the manufacturer's instructions for use, RNA is isolated from the cells using the RNA Extraction Kit (Genaid), and then the RNA is complemented with deoxyribonucleic acid by using the reverse transcriptase SuperScript® III Reverse Transcriptase (Invitrogen) at 37°C. cDNA), followed by Q-PCR kit (KAPA CYBR FAST qPCR Kit (2X), KAPA Biosystems), forward primer 5'-GATACCCCTTTGACGGTAAGGA-3' (SEQ ID NO: 1) against MMP2 gene (SEQ ID NO: 1) and reverse primer 5' Gene expression data was obtained by performing PCR on the cDNA using -CCTTCTCCCAAGGTCCATAGC-3' (SEQ ID NO: 2), and a polymerase chain reactor (StepOnePlus™ Real-Time PCR Systems).

Example 1

Preparation of mimosa extract

First, the mimosa is washed and dried naturally, and then the mimosa is ground using a homogenizer. Subsequently, the mimosa homogenate is extracted with a solvent of water, alcohols, or an alcohol-water mixture, and the mixed liquid-solid ratio of the solvent and the mimosa homogenate is 5 to 20: 1 to 5. The extraction temperature is between 50°C and 100°C, preferably between 75°C and 95°C. The extraction time in this example is 0.5 to 2 hours.

The mimosa extract obtained through the extraction step is cooled to room temperature, and then filtered through a 400 mesh filter screen to remove residual solids. Further, the mimosa extract after filtration is further concentrated in vacuo at 45°C to 70°C to obtain a concentrated product. In order to obtain a solid mimosa extract, a solvent is removed from the mimosa extract that has been subjected to vacuum concentration using a spray drying method to obtain a mimosa extract powder.

Example 2

Effects of Mimosa Extract to Improve Fibroblast Collagen Production

In order to verify the effect of the mimosa extract according to the present invention on the collagen production of skin cells, this example analyzes changes in collagen production after treating human skin fibroblast CCD-966SK with mimosa water extract through a collagen content experiment. Did. First, human skin fibroblasts were inoculated into each well of a 24-well plate containing 500 μL of cell medium at a density of 2×10 ⁴ cells/well, incubated at 37° C. for 24 hours, and then the cell medium was removed and PBS solution. Wash cells using. Subsequently, 500 μL, 1 mg/mL of mimosa water extract and 500 μL of cell medium not containing FBS were added to the cells of each well, and cells were also treated using only 500 μL of FBS-free cell medium. Used as a control. Cells of the two groups were incubated at 37° C. for 48 hours, and then cell media was collected from each well to measure the collagen content therein.

Figure 1 shows the relative production of collagen in human skin fibroblasts and control cells treated with mimosa extract according to the present invention. As shown in Figure 1, compared to the human skin fibroblasts of the control group, in human skin fibroblasts treated with mimosa extract, the collagen content in the cell medium was clearly increased by about 8.3 times. As can be seen from the above results, the mimosa extract has an effect of improving collagen production of skin fibroblasts.

Example 3

Effect of Mimosa Extract on Inhibiting Gene Expression in Collagen Decomposition

Furthermore, in order to explain the regulation action of the mimosa extract according to the present invention for gene expression of collagen degrading enzyme, this embodiment treats human skin fibroblast CCD-966SK (BCRC 60153) with mimosa water extract through Q-PCR. Then, changes in expression levels of genes related to collagen degradation were measured. First, human skin fibroblasts were inoculated into each well of a 6-well plate containing 2 mL of cell medium at a density of 1.5×10 ⁵ cells/well, and then cultured at 37°C. Subsequently, the cells were treated with mimosa water extract at 0.5 mg/mL or 1 mg/mL for 24 hours, and then the treated cells were collected to conduct Q-PCR. Human skin fibroblasts of the control were treated with a cell medium containing no mimosa water extract.

The relative expression level of the matrix metalloproteinase 2 (MMP2) gene in the human skin fibroblasts is as shown in FIG. 2, and the relative expression level of the vertical axis in the figure is a multiple relative to the RNA expression level of the MMP2 gene in the control cells. Means MMP2 is also called type IV collagenase, which is synthesized in cells and then secreted into an extracellular matrix to function to break down type 4 collagen in the basement membrane below the epithelial tissue. As shown in FIG. 2, when human skin fibroblasts are treated with a mimosa extract having a concentration of 0.5 mg/mL or more, gene expression of MMP2 can be significantly lowered. When the cells were treated with the mimosa extract at 0.5 mg/mL, the MMP2 gene expression was 46% of the control cells. By the above results, it can be seen that the mimosa extract according to the present invention can suppress the gene expression of MMP2 in skin fibroblasts and thus has an inhibitory effect on collagen degradation in skin.

As described above, the mimosa extract obtained by extracting a mixture of water, alcohols, and alcohol-water according to the present invention suppresses the gene expression of MMP2 in skin fibroblasts to reduce the degradation of type 4 collagen in the extracellular matrix. Can finally achieve the effect of increasing the collagen content in the skin. Therefore, the mimosa extract according to the present invention can be used for the preparation of a composition that inhibits the expression of MMP2 gene and the degradation of collagen, and the composition may be in powder form, granule form, liquid form, gel form or paste form, and food It can be prepared as a beverage, drug, reagent, or nutritional supplement, and can be used on an individual by oral administration or application to the skin.

SEQUENCE LISTING <110> TCI Co., Ltd <120> Use of Mimosa pudica extracts for manufacture of composition for inhibiting MMP2 gene expression and collagen degradation <130> 106B0169-I1 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 22 <212> DNA <213> artificial sequences <220> <223> PCR primer <400> 1 gatacccctt tgacggtaag ga 22 <210> 2 <211> 21 <212> DNA <213> artificial sequences <220> <223> PCR primer <400> 2 ccttctccca aggtccatag c 21

Claims (13)

A cosmetic composition for inhibiting the expression of a matrix metalloproteinase 2: MMP2 gene, comprising a mimosa extract obtained by extracting Mimosa pudica using a solvent,
The solvent is water,
The liquid-solid ratio of the solvent and the mimosa is 5 to 20:1 to 5 in mass ratio,
The extraction proceeds at a temperature between 50 ℃ to 100 ℃,
The mimosa extract is a mimosa-water extract, characterized in that the concentration is 0.5 to 1 mg/mL, composition. delete delete delete delete The composition according to claim 1, wherein the composition is administered by oral administration or applied to the skin. A cosmetic composition for inhibiting collagen decomposition, comprising a mimosa extract obtained by extracting mimosa pudica using a solvent,
The solvent is water,
The liquid-solid ratio of the solvent and the mimosa is 5 to 20:1 to 5 in mass ratio,
The extraction proceeds at a temperature between 50 ℃ to 100 ℃,
The mimosa extract is a mimosa-water extract, characterized in that the concentration is 0.5 to 1 mg/mL, composition. delete delete delete delete The composition according to claim 7, wherein the collagen is type 4 collagen. The composition according to claim 7, wherein the composition is administered by oral administration or applied to the skin.

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