TWI718389B - Use of 3'-hydroxygenistein for anti-photoaging - Google Patents

Abstract

Use of 3’-hydroxygenistein is disclosed. The 3’-hydroxygenistein has an anti-aging capability. Therefore the 3’-hydroxygenistein can be used to manufacture compositions for anti-aging.

Description

Use of 3'-hydroxygenistein for anti-photoaging

The present invention relates to the use of 3'-hydroxy genistein, in particular to the use of 3'-hydroxy genistein for anti-aging.

In biology and medicine, aging refers to the phenomenon that the physiological state of an organism deteriorates over time. The aging of the skin can be caused by internal and external influences. The internal influence is intrinsic aging, and The external influence is mainly due to long-term exposure to the sun. The ultraviolet (UV) and infrared (IR) in the sun damage the skin and cause skin aging, also known as photoaging.

The typical clinical manifestations of skin photoaging are roughness, thickening, dryness, sagging skin, deepening and thickening of wrinkles, local hyperpigmentation or telangiectasia and other skin conditions, and even various benign or malignant tumors (such as Solar keratosis, squamous cell carcinoma, malignant melanoma, etc.).

The symptoms of skin photoaging are closely related to the changes in the dermal connective tissue and the changes in the microstructure. These changes include the decomposition of enzymes and the reduction of newly produced collagen, which makes the skin wrinkle earlier. In addition, even a small amount of ultraviolet rays can damage the skin's main structural protein-collagen fibers, causing some abnormal elastin to accumulate, causing the skin to lose its elasticity. The changes in the matrix of hyaluronic acid and proteoglycan will also reduce the water content of the cells and allow the cells to relax.

One aspect of the present invention is to provide a use of 3'-hydroxygenistein (3'-hydroxygenistein), which is used to prepare an anti-aging composition.

According to the use of the aforementioned 3'-hydroxygenistein, the anti-aging composition can be a matrix metalloproteinase-1 (MMP-1) inhibitor.

According to the use of the aforementioned 3'-hydroxygenistein, the anti-aging composition can be used to prevent skin photoaging caused by exposure to ultraviolet radiation. The anti-aging composition can also be used to reduce skin photoaging. The skin photoaging condition can be selected from the group consisting of wrinkles, pigmentation and/or hyperpigmentation, rough or leathery skin, sagging or sagging, yellowing, dryness, and skin vegetation.

According to the use of the aforementioned 3'-hydroxygenistein, the effective dose of the anti-aging composition can be from 6.25 μg/mL to 2000 μg/mL. Preferably, the effective dose of the anti-aging composition may be 18 μg/mL to 1000 μg/mL. More preferably, the effective dose of the anti-aging composition is 25 μg/mL to 200 μg/mL.

According to the use of the aforementioned 3'-hydroxy genistein, the anti-aging composition can be a liquid, an emulsion, an ointment or a powder.

According to the use of the aforementioned 3'-hydroxygenistein, the anti-aging composition can be a pharmaceutical composition, a cosmetic composition or a health food composition.

Thereby, the 3'-hydroxygenistein of the present invention has the ability to inhibit matrix metalloproteinase-1, thereby achieving anti-aging effects, especially preventing or reducing skin photoaging. Therefore, 3'-hydroxygenistein can be used to prepare anti-aging compositions, which can be pharmaceutical compositions, cosmetic compositions or health food compositions, and have the potential to be used in the biomedical and health care market.

The above content of the invention aims to provide a simplified summary of the content of the disclosure, so that readers have a basic understanding of the content of the disclosure. This summary is not a complete summary of the present disclosure, and its intention is not to point out important/key elements of the embodiments of the present invention or to define the scope of the present invention.

In order to make the above and other objectives, features, advantages and embodiments of the present invention more comprehensible, the description of the accompanying drawings is as follows: Figure 1 shows the cell survival rate of 3'-hydroxygenistein on human fibroblasts Figure 2 shows the results of the analysis of effects; and Figure 2 shows the results of the analysis of 3'-hydroxygenistein inhibiting the activity of matrix metalloproteinase-1.

The disclosure of this specification proposes the novel use of 3'-hydroxy genistein, especially the novel application of 3'-hydroxy genistein for anti-aging. To evaluate the anti-aging effect of 3'-hydroxygenistein with cell in vitro tests, and to further verify that 3'-hydroxygenistein can inhibit the activity of matrix metalloproteinase-1 (MMP-1). Achieve anti-aging effect. The 3'-hydroxygenistein proposed in the disclosure of this specification is a potential anti-aging substance, especially as a substance that inhibits matrix metalloproteinase-1, and can be used to prepare a pre-anti-aging pharmaceutical composition.

The following specific test examples are used to further illustrate the present invention, so as to benefit those who are generally knowledgeable in the technical field to which the present invention belongs, and can fully utilize and practice the present invention without excessive interpretation. These test examples should not be regarded as It limits the scope of the present invention, but is used to illustrate how to implement the materials and methods of the present invention.

<Test Example> 1. The cytotoxic effect of 3’-hydroxygenistein on human fibroblasts

3’-羥基染料木黃酮的製備方式有很多種，例如利用生物轉換作用(biotransformation)製備而得，但本發明在此並不加以限定其製備方式，舉凡涉及利用3’-羥基染料木黃酮用於製備抗老化之組合物，應視為本發明所請之範圍內。 3'-Hydroxygenistein is a derivative of genistein. Genistein is one of the main active factors of soy isoflavones. It has a variety of physiological functions. The chemical structure of 3'-hydroxygenistein is as follows:

There are many ways to prepare 3'-hydroxy genistein, such as biotransformation. However, the present invention does not limit its preparation method here. For example, it involves the use of 3'-hydroxy genistein. The preparation of anti-aging compositions should be regarded as within the scope of the present invention.

In order to determine the safety and safe dose of 3'-hydroxygenistein to normal cells, human fibroblasts were tested for cell survival with different doses of 3'-hydroxygenistein. The cell line used in this experiment was the human fibroblast cell line Hs68, which was purchased from the Bioresource Collection and Research Center (BCRC) of the Food Industry Development Institute, and the number was BCRC 60038. The cell culture medium is DMEM (Dulbecco's modified Eagle's medium), which contains 10% fetal bovine serum, 100μg/ml streptomycin and 100unit/ml penicillin. The Hs68 cells were cultured in an incubator with the aforementioned cell culture medium to maintain 5% CO ₂ and a constant temperature of 37° C. The cells were subcultured every 3 days.

When measuring cell viability, first ^{inoculate Hs68 cells in a 96-well cell analysis dish at a density of 1×10 4} cells/well, and incubate the cells at 37°C and 5% CO ₂ until the cells adhere to the wall the next day. , Remove the original cell culture fluid, and then add 3'-hydroxygenistein serially diluted with the cell culture fluid to the 96-well cell analysis dish as the experimental group. The final concentration of 3'-hydroxygenistein in the experimental group is 31.25. μg/mL, 62.5μg/mL, 125μg/mL, 250μg/mL, 500μg/mL, 1000μg/mL and 2000μg/mL, 3'-hydroxygenistein samples of each concentration are subjected to 3 repeated tests, and the test also includes Control group of untreated 3'-hydroxygenistein. And use alamarBlue® reagent (Thermo) to detect the cell viability of Hs68 cells, and then use FlexStation 3 to read the values and analyze.

Please refer to Figure 1, which is the analysis result of the effect of 3'-hydroxygenistein on the cell survival rate of human fibroblasts. The cell survival rate is based on the cell survival rate of the control group as 100%. The results in Figure 1 show that the cell survival rate of Hs68 cells in the experimental group is greater than 100%, indicating that 3'-hydroxygenistein is not cytotoxic to Hs68 cells, and its safe dose can reach 2000 μg/mL.

2. The anti-aging effect of 3’-hydroxygenistein

Matrix metalloproteinase-1 is a type of collagen-degrading enzyme, and it is an important factor for regulating photoaging. When fibroblasts are stimulated by ultraviolet radiation from sunlight, they will trigger the cell membrane to produce lipid peroxidation. Lipid peroxidation promotes the mass production of matrix metalloproteinase-1, which in turn degrades the extracellular matrix (ECM) and makes the human body The collagen in the dermal tissue of the skin is degraded, which affects the contraction and elasticity of the skin, and induces premature aging of the skin.

Therefore, the anti-aging analysis test of this test example first treated human fibroblast cell lines with different doses of 3'-hydroxy genistein, and then analyzed the 3'-hydroxy group by enzyme-linked immunosorbent assay (ELISA). The inhibitory effect of genistein on matrix metalloproteinase-1 and cell survival test.

When conducting anti-aging analysis test, first ^{inoculate Hs68 cells in a 96-well cell analysis dish at a density of 1×10 4} cells/well, and incubate the cells at 37°C and 5% CO ₂ until the cells adhere to the wall the next day. Remove the original cell culture fluid, and then add 3'-hydroxygenistein serially diluted with the cell culture fluid to the 96-well cell analysis dish as the experimental group. The final concentration of 3'-hydroxygenistein in the experimental group is 6.25μg. /mL, 12.5μg/mL, 25μg/mL, 50μg/mL, 100μg/mL and 200μg/mL, 3'-hydroxygenistein samples of each concentration are subjected to 3 repeated tests, and the test also includes untreated 3'- The control group of hydroxygenistein. After Hs68 cells were treated with 3'-hydroxygenistein for 6 hours, 10ng/mL tumor necrosis factor-α (Tumor necrosis factor-α, TNF-α) was added to each well of the 96-well cell analysis plate to stimulate Hs68 cells Produce matrix metalloproteinase-1. The Hs68 cells after reprocessing TNF-α were cultured at 37°C and 5% CO _{2 for} 18 hours, and then the supernatants of the control group and each experimental group were collected. The supernatant was pre-coated with matrix metalloproteinase-1 antibody for enzyme-binding immunosorbent analysis to detect the content of matrix metalloproteinase-1 in the supernatant of the control group and each experimental group. And use alamarBlue® reagent (Thermo) to detect the cell viability of Hs68 cells, and then use FlexStation 3 to read the values and analyze.

Please refer to Figure 2, which shows the analysis results of 3'-hydroxygenistein inhibiting the activity of matrix metalloproteinase-1. It can be seen from the results in Figure 2 that the cell survival rate of Hs68 cells in the control group and the experimental group is greater than 80%, indicating that 3'-hydroxygenistein is not cytotoxic to Hs68 cells. In addition, it can be seen from the control group that the matrix metalloproteinase-1 produced by Hs68 cells without TNF-α is about 1.3 ng/mL, while the matrix metalloproteinase-1 produced by Hs68 cells with TNF-α is about 6.7 ng/mL. mL, showing that processing TNF-α does make Hs68 cells produce a large amount of matrix metalloproteinase-1. In the part of the experimental group, it can be seen that the content of matrix metalloproteinase-1 produced by Hs68 cells with or without TNF-α is reduced. In the Hs68 cell portion of the experimental group treated with TNF-α, the content of matrix metalloproteinase-1 produced by the treatment of 3'-hydroxygenistein from low to high (6.25μg/mL to 200μg/mL) was 4.96ng respectively. /mL, 4.25ng/mL, 3.15ng/mL, 2.34ng/mL, 1.56ng/mL and 0.88ng/mL. In the Hs68 cell portion of the experimental group that was not treated with TNF-α, the amount of matrix metalloproteinase-1 produced by it was from low to high (6.25μg/mL to 200μg/mL) depending on the concentration of treated 3'-hydroxygenistein. 1.01ng/mL, 0.96ng/mL, 0.92ng/mL, 0.84ng/mL, 0.93ng/mL and 0.68ng/mL. Please refer to the following table 1 again for the calculation of the inhibition rate of 3'-hydroxygenistein on MMP-1 based on the above data. The calculation formula of the inhibition rate is as follows:

The results in Table 1 show that treatment of 3'-hydroxygenistein has a dose-dependent inhibition rate of matrix metalloproteinase-1 in Hs68 cells. In the experimental group without treatment of TNF-α, the application concentration is 6.25μg/mL to 200μg /mL of 3'-hydroxygenistein can inhibit the production of matrix metalloproteinase-1 in Hs68 cells from 22.4% to 47.9%. In the experimental group treated with TNF-α, 3'-hydroxygenistein with a concentration of 6.25μg/mL to 200μg/mL can inhibit the production of matrix metalloproteinase-1 in Hs68 cells by 25.5% to 86.9%. And the IC50 of 3'-hydroxygenistein for inducible matrix metalloproteinase-1 is 18.53±0.90μg/mL (77.44±3.76μM), which shows that 3'-hydroxygenistein is for endogenous Both the sexual matrix metalloproteinase-1 and the inducible matrix metalloproteinase-1 have a good inhibitory effect.

According to the above test results, 3'-hydroxygenistein has a good inhibitory effect on matrix metalloproteinase-1, so it can be used as an anti-aging composition, especially a matrix metalloproteinase-1 inhibitor, and can be used to prevent An anti-aging composition for skin photoaging that occurs due to exposure to ultraviolet radiation, or can be used to reduce skin photoaging, and the skin photoaging can be selected from wrinkles, stains and/or Hyperpigmentation, rough or leathery skin, sagging or sagging, yellowing, dryness and skin vegetation.

The effective dose of the anti-aging composition may be 6.25 μg/mL to 2000 μg/mL. Preferably, the effective dose of the anti-aging composition may be 18 μg/mL to 1000 μg/mL. More preferably, the effective dose of the anti-aging composition is 25 μg/mL to 200 μg/mL.

The anti-aging composition can be a liquid, an emulsion, an ointment or a powder, and the anti-aging composition can be a pharmaceutical composition, a cosmetic composition or a health food composition.

When the anti-aging composition is a pharmaceutical composition, it can be prepared according to the accepted pharmaceutical manufacturing process, and may further include one or more pharmaceutically acceptable carriers. The above-mentioned pharmaceutically acceptable carriers may include, but not Limited to solvents, dispersion mediums, coatings, antibacterial and antifungal agents, and first-class osmotic pressure and absorption delaying agents that are compatible with pharmaceutical administration. For different modes of administration, general methods can be used to formulate the pharmaceutical composition into a dosage form. For example, the dosage form for oral administration may include, but is not limited to, tablets, capsules, emulsions, aqueous suspensions, dispersions and solutions.

When the anti-aging composition is a cosmetic composition, it may further include a generally widely used cosmetically acceptable adjuvant (cosmetically acceptable adjuvant), such as one or more selected from solvents, gelling agents, active agents, preservatives Adjuvants, antioxidants, screening agents, chelating agents, surface active agents, thickening agents, fragrances and odor absorbers, and the selection of adjuvants is for those skilled in the art Within the scope of professional common sense and routine technology.

In addition, the anti-aging composition can be applied as an external application. In the case of external application, a vehicle can be used as a diluent, dispersant or carrier for the active ingredient. Vehicles may include materials commonly used in skin care products, such as water, liquid or solid softeners, silicone oils, emulsifiers, solvents, humectants, thickeners, powders, propellants, and the like. The vehicle may constitute the rest of the composition in the absence of other adjuvants.

In summary, the 3'-hydroxygenistein of the present invention has an excellent ability to inhibit matrix metalloproteinase-1, thereby achieving anti-aging effects, especially preventing or reducing skin photoaging. Therefore, 3'-hydroxy dye wood flavonoids can be used to prepare anti-aging compositions, which can be pharmaceutical compositions, cosmetic compositions or health food compositions, and have the potential to be used in the biomedical and health care market.

Although the present invention has been disclosed in the above embodiments, it is not intended to limit the present invention. Anyone familiar with the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection of the present invention The scope shall be subject to those defined by the attached patent scope.

Claims (9)

A use of 3'-hydroxygenistein (3'-hydroxygenistein), which is used to prepare an anti-photoaging composition, wherein the photoaging is a skin photoaging, wherein the skin photoaging condition is selected Free wrinkles, rough or leathery skin, sagging or sagging, yellowing, dryness, and skin vegetation. The use of 3'-hydroxygenistein as described in item 1 of the patent application, wherein the anti-photoaging composition is a matrix metalloproteinase-1 (Matrix Metalloproteinase-1, MMP-1) inhibitor. The use of 3'-hydroxygenistein as described in item 2 of the scope of patent application, wherein the anti-photoaging composition is used to prevent a skin photoaging condition caused by exposure to ultraviolet radiation. The use of 3'-hydroxygenistein as described in item 2 of the scope of patent application, wherein the anti-photoaging composition is used to reduce a skin's photoaging condition. The use of 3'-hydroxygenistein as described in item 1 of the scope of patent application, wherein the effective dose of the anti-photoaging composition is from 6.25 μg/mL to 2000 μg/mL. The use of 3'-hydroxygenistein as described in item 5 of the scope of patent application, wherein the effective dose of the anti-photoaging composition is 18 μg/mL to 1000 μg/mL. The use of 3'-hydroxygenistein as described in item 6 of the scope of patent application, wherein the effective dose of the anti-photoaging composition is 25 μg/mL to 200 μg/mL. The use of 3'-hydroxygenistein as described in item 1 of the scope of patent application, wherein the anti-photoaging composition is a liquid, an emulsion, an ointment or a powder. The use of 3'-hydroxygenistein as described in item 1 of the scope of patent application, wherein the anti-photoaging composition is a pharmaceutical composition or a cosmetic composition.

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