TWI636790B - Use of mimosa pudica extracts for manufacture of composition for inhibiting mmp2 gene expression and collagen degradation - Google Patents

Abstract

The invention provides the use of a couple of grass extracts for preparing a composition for inhibiting the expression of matrix metalloproteinase 2 (MMP2) gene, and a use of a couples grass extract for preparing a composition for inhibiting the degradation of collagen. The extract of the couple's grass of the present invention is prepared by extracting the couple's grass with water, alcohol, or an alcohol-water mixture as a solvent. The marjoram extract reduces the decomposition of collagen in the skin by inhibiting the gene expression of MMP2 in the skin fibroblasts, thereby helping to improve skin elasticity and firmness, reduce skin fine lines and wrinkles, and delay skin aging.

Description

Use of marjoram grass extract for preparing composition for inhibiting MMP2 gene expression and collagen degradation

The present invention relates to the use of an extract of a couple of grasses, in particular to the use of an extract of a couple of grasses for preparing a composition that inhibits the expression of matrix metalloproteinase 2 (MMP2) gene and inhibits the degradation of collagen.

Collagen (collagen) is a highly abundant protein in animals, accounting for about 25% of the total protein in the body. Collagen is mainly distributed in the extracellular matrix of connective tissue in animals, such as ligaments, tendons, cartilage, and cornea of the eye. It is the most important protein in connective tissue, accounting for about 20% to 30% of connective tissue. Collagen has a fibrous structure and can provide the mechanical support and strength required for connective tissue. It is also related to blood coagulation, wound healing, and tissue repair, making it a very important structural protein. Collagen in the human body can be divided into different types due to the difference in polypeptide composition. Among them, type I, type III, and type IV collagen are most relevant to the structure of the skin. The dermal layer contains type 1 and type 3 collagens, which are related to the skin's moisture retention and elasticity, and the basement membrane below the epithelial tissue contains type 4 collagens, which are related to the skin's water transport and nutrient maintenance.

With age and long-term UV exposure, collagen in the skin will be lost and the skin's water content will decrease. The dry state of the skin will further cause poor skin elasticity, wrinkle formation, and enlarged pores. Therefore, efforts to replenish skin collagen, improve collagen synthesis, and reduce collagen degradation have become the focus of research on slowing skin aging and maintaining skin health. Due to the barrier effect of the epidermal layer of the skin, applying skin care products containing collagen directly to the skin surface cannot effectively supplement the deep collagen of the skin. On the other hand, if collagen is taken orally, The original protein is broken down into small molecules of amino acids in the digestive tract. After obtaining these amino acids in various parts of the human body, it may not necessarily synthesize the collagen required. Therefore, the effect of oral collagen to enhance the synthesis of skin collagen is limited.

In view of this, it is necessary to develop a novel composition that can effectively inhibit the breakdown of collagen to reduce the loss of collagen and avoid skin aging.

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

Another object of the present invention is to provide a use of the aforesaid extract of a couple of grasses for preparing a composition for inhibiting the degradation of collagen, wherein the extract of a couple of grasses is obtained by extracting a couple of grasses with a solvent.

In one embodiment of the present invention, the solvent is water, alcohols, or a mixture of alcohol and water, and the liquid-to-solid ratio of the solvent to the couple's grass is 5-20: 1-5, and the extraction system is at 50 ° C to 100 ° C. get on.

In one embodiment of the present invention, the extract of the couple's grass is a water extract of the couple's grass, and its concentration is at least 0.5 mg / mL.

In one embodiment of the present invention, the collagen is type IV collagen.

In the present invention, the extract of Marjoram humilis extracted with water, alcohols, or a mixture of alcohol and water as a solvent can reduce the degradation of type IV collagen in the extracellular matrix by inhibiting the gene expression of MMP2 in skin fibroblasts, and finally achieve The effect of increasing the collagen content in the skin, thus helping to improve skin elasticity and firmness, reduce skin fine lines and wrinkles, and delay skin aging. Therefore, the extract of the couple's grass of the present invention can be used to prepare a composition for inhibiting the expression of the MMP2 gene and inhibiting the decomposition of collagen. The composition can be powder, granule, liquid, gel or paste, and can be made into food and drink , Medicines, reagents or nutritional supplements, given to a body by oral administration, skin application, etc.

The embodiments of the present invention will be further described below with reference to the drawings. The examples listed below are intended to clarify the features and applications of the invention, but not to limit the scope of the invention. Anyone skilled in the art will not depart from it. Within the spirit and scope of the present invention, some modifications and retouching can be done. Therefore, the protection scope of the present invention shall be determined by the scope of the attached patent application.

FIG. 1 shows the relative collagen production of human skin fibroblasts and human skin fibroblasts in the control group treated with the extract of the couple's grass.

FIG. 2 shows the relative expression of the MMP2 gene in human skin fibroblasts treated with the extract of Acanthopanax splendens.

The invention provides the use of a couple's grass extract for preparing a composition for inhibiting the degradation of collagen. The extract of the couple's grass of the present invention is obtained by extracting a plant of the couple's grass with a solvent, wherein the solvent is water, alcohol, or a mixture of alcohol and water, and the liquid-solid ratio of the solvent to the couple's grass is 5-20: 1 to 5, and the extraction is performed at 50 ° C to 100 ° C. Through the collagen content test and the expression analysis of genes related to collagen degradation, the extract of the couple's grass of the present invention was confirmed to increase the collagen production of human skin fibroblasts, and the increase in the production was related to the decrease in the expression of the MMP2 gene. Therefore, the extract of Leymus chinensis according to the present invention can also be used to prepare a composition for inhibiting the expression of the MMP2 gene.

definition

The values used in this article are approximate. All experimental data are shown in the range of 20%, preferably in the range of 10%, and most preferably in the range of 5%.

Materials and Methods material

Eagle's Minimum Essential Medium (MEM medium), Fetal bovine serum (FBS), non-essential amino acids, sodium bicarbonate, sodium pyruvate, purchased from Gibco with Earle's balanced salt solution, And phosphate buffered saline (PBS buffer solution).

Cell culture

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

Collagen content test

In the present invention, a soluble collagen analysis kit (Sircol Soluble Collagen Assay Kit, Bicolor) is used to determine the content of collagen secreted by cells. The steps are briefly described below. Take 1000 μL The cell culture medium was mixed with 200 μL of a collagen separation / concentration reagent, and after being inverted at 4 ° C. for a period of time, the mixture was centrifuged at 12000 rpm for 10 minutes, and the supernatant was removed. Next, the remaining precipitate was mixed with 1000 μL of Sircol dye for 30 minutes, and the dye mixture was centrifuged at 12000 rpm for 10 minutes to form a collagen precipitate. To remove the supernatant, first wash the collagen precipitate with 750 μL of acidic salt solution and remove excess liquid, add 250 μL of alkaline reagent to dissolve the collagen precipitate, and then use an enzyme immunoassay analyzer (ELISA reader, BioTek) The absorbance of the collagen solution at a wavelength of 555 nm was measured. By comparing the standard curve of the water-soluble collagen standard, the collagen content in the cell culture medium to be measured can be calculated. Statistical analysis is performed using Student's t-test of Excel software.

Gene expression analysis

In the present invention, the quantitative polymerase chain reaction (Q-PCR) is used to determine the gene expression of enzymes related to the degradation of collagen in cells. The steps are briefly described below. According to the manufacturer's instructions, RNA was isolated from the cells using the RNA Extraction Kit (Geneaid), and the RNA was reverse transcribed into complementary deoxyribose at 37 ° C using the reverse transcriptase SuperScript® III Reverse Transcriptase (Invitrogen). Nucleic acid (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 and anti- This primer was 5′-CCTTCTCCCAAGGTCCATAGC-3 ′ (SEQ ID NO: 2) and a polymerase chain reaction system (StepOnePlus ^™ Real-Time PCR Systems) was used to perform PCR on the cDNA to obtain gene expression data.

Example 1 Preparation of Marjoram grass extract

First, the couple grass was washed and dried, and the couple grass was ground with a homogenizer. Secondly, water, alcohols, or a mixture of alcohol and water are used as a solvent to extract the homogenate of the husband and wife, and the liquid-solid ratio of the solvent and the homogenate of the husband and wife is 5 ~ 20: 1 ~ 5. The extraction temperature is between 50 ° C and 100 ° C, preferably between 75 ° C and 95 ° C. In this embodiment, the extraction time is 0.5 to 2 hours.

After cooling down to room temperature, the extract of the couple's grass obtained through the above-mentioned extraction step can be filtered through a 400 mesh filter to remove residual solids. The filtered Marjoram grass extract can be further concentrated under reduced pressure at 45 ° C to 70 ° C to obtain a concentrated product. In order to obtain The solvent extract was removed by spray-drying the above-mentioned condensed fragrant grass extract under reduced pressure, thereby obtaining a fragrant grass extract powder.

Example 2 Marjoram extract improves fibroblast collagen production

In order to test the effect of the extract of the plant of the present invention on the collagen production of skin cells, this example uses a collagen content test to analyze the human skin fibroblast CCD-966SK treated with the water extract of the plant of the plant and its collagen production. Volume change. First, human skin fibroblasts were seeded at 2 × 10 ⁴ cells / well into each well of a 24-well plate containing 500 μL of cell culture medium, and after being cultured at 37 ° C. for 24 hours, the cell culture medium was removed and the cells were washed with a PBS solution. Secondly, 500 μL, 1 mg / mL water extract of Amaryllis and 500 μL of FBS-free cell culture medium were added to each well cell, or the cells were treated with only 500 μL of FBS-free cell culture medium as a control group. After the two groups of cells were cultured at 37 ° C for 48 hours, the cell culture medium was collected from each well, and the collagen content was measured.

FIG. 1 shows the relative collagen production of human skin fibroblasts and cells in the control group treated with the extract of the couple's grass. According to FIG. 1, compared with the control group of human skin fibroblasts, the application of the extract of the grass to the human skin fibroblasts significantly increased the collagen content in the cell culture medium by about 8.3 times. This result shows that the extract of the marjoram grass of the present invention has the effect of increasing the collagen production of skin fibroblasts.

Example 3 Expression of Marjoram grass extract inhibiting genes related to collagen degradation

In order to further explore the regulatory effect of the extract of the plant of the present invention on the collagen-degrading enzyme, Q-PCR was used to determine the human skin fibroblasts CCD-966SK (BCRC 60153) treated with the water extract of the plant. Changes in the expression of collagen-decomposition-related genes. First, human skin fibroblasts were seeded at 1.5 × 10 ⁵ cells / well into each well of a 6-well plate containing 2 mL of cell culture medium, and cultured at 37 ° C. Next, the cells were treated with 0.5 mg / mL or 1 mg / mL water extract of Amaryllis for 24 hours, and the treated cells were collected for Q-PCR. The human skin fibroblast cell line of the control group was treated with a cell culture medium that did not contain a water extract of the marjoram grass.

The relative expression amount of the matrix metalloproteinase 2 (MMP2) gene in the human skin fibroblasts is shown in FIG. 2. The relative expression amount of the gene in the vertical axis represents a multiple of the expression amount of the RNA relative to the MMP2 gene in the control group cells. MMP2 is also known as type IV collagenase collagenase), which is synthesized by cells and secreted into the extracellular matrix, and its functions include breaking down type IV collagen in the basement membrane below the epithelial tissue. According to FIG. 2, the administration of the extract of Aquilegia japonica on human skin fibroblasts at a concentration of 0.5 mg / mL or more significantly reduced the gene expression of MMP2. When the aforesaid cells were treated with 0.5 mg / mL extracts of Aquilegia japonica, the expression of MMP2 gene was about 46% of the cells in the control group. This result indicates that the extract of the couple's grass can inhibit the gene expression of MMP2 in skin fibroblasts, and therefore has an inhibitory effect on the degradation of collagen in the skin.

To sum up, the water extract, the water extract, the alcohol extract, or the alcohol-water mixture extracted by the present invention can reduce the MMP2 gene expression in the skin fibroblast cells and reduce the type IV collagen in the extracellular matrix. Degradation, and ultimately achieve the effect of increasing collagen content in the skin. Therefore, the extract of the marjoram grass of the present invention can be used to prepare a composition for inhibiting the expression of MMP2 gene and inhibiting the degradation of collagen. The composition can be powder, granular, liquid, gel or paste, and can be made into food, Drinks, medicines, reagents or nutritional supplements are administered to a body by oral administration, skin application, and the like.

<110> Dajiang Biomedical Co., Ltd.

<120> Use of marjoram grass extract for preparing composition for inhibiting MMP2 gene expression and collagen degradation

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Claims (3)

A mangosteen extract is used for preparing a composition for inhibiting the expression of matrix metalloproteinase 2 (MMP2) gene, wherein the mangosteen extract is a solvent selected from water, alcohols, and a mixture of alcohol and water at 50 ° C. It is obtained by extracting a couple grass at 100 ° C, and the liquid-solid ratio of the solvent to the couple grass is 5 ~ 20: 1 ~ 5. The use according to item 1 of the scope of the patent application, wherein the extract of the couple's grass is a water extract of the couple's grass and its concentration is at least 0.5 mg / mL. The use according to item 1 of the scope of patent application, wherein the composition is administered orally or by skin application.