CN107028782B - Application of isoliquiritigenin or isoliquiritin in preparation of high-safety anti-aging cosmetic - Google Patents
Application of isoliquiritigenin or isoliquiritin in preparation of high-safety anti-aging cosmetic Download PDFInfo
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- CN107028782B CN107028782B CN201610081250.1A CN201610081250A CN107028782B CN 107028782 B CN107028782 B CN 107028782B CN 201610081250 A CN201610081250 A CN 201610081250A CN 107028782 B CN107028782 B CN 107028782B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
- A61K8/35—Ketones, e.g. benzophenone
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/60—Sugars; Derivatives thereof
- A61K8/602—Glycosides, e.g. rutin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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Abstract
The invention discloses an application of isoliquiritigenin or isoliquiritin in preparing anti-aging cosmetics with high safety. According to the invention, the isoliquiritigenin or isoliquiritin is found to have the effects of improving the expression level of the alpha 1 chain (Col1A1) of the type I collagen in human skin fibroblasts, reducing the expression level of matrix metalloproteinase 1(MMP1) and promoting the expression level of sirtuin 1(Sirt1), so that the isoliquiritin or isoliquiritin can inhibit the apoptosis of the human skin fibroblasts, promote the synthesis of collagen in the human skin fibroblasts and further achieve the anti-aging purpose. And has low toxicity to human skin fibroblasts. The research shows that isoliquiritigenin and isoliquiritin have important application value in preparing anti-aging cosmetics with high safety.
Description
Technical Field
The invention relates to a new application of isoliquiritigenin or isoliquiritin, in particular to an application of isoliquiritigenin or isoliquiritin in inhibiting apoptosis of human skin fibroblasts and promoting synthesis of collagen in human skin fibroblasts, belonging to the technical field of cosmetics.
Background
The Glycyrrhiza uralensis Fisch is called as whitening queen, is widely applied to cosmetics and has great market demand, and can inhibit tyrosinase, remove free radicals and inhibit histamine release, so that the effects of whitening, resisting aging and inflammation are achieved, and the problems of rough skin, water shortage and the like can be solved. Liquorice whitening is reported in many foreign whitening cosmetic patents, and liquorice extract is often directly added. Many cosmetic products with alleged whitening effect contain licorice extract. Since licorice extract is generally dark in color, its use amount in cosmetics is limited. Therefore, the crude extract has a great limitation in the application to cosmetics. The liquorice comprises the following main components: polysaccharides, triterpenes, flavonoids, a small amount of alkaloid, lignin and coumarin. Among them, 106 flavonoids and 73 aglycones were reported. The compounds separated from liquorice at present comprise glycyrrhizin, glycyrrhetinic acid, liquiritin, Glabridin, isoliquiritin, neoliquiritin, neoisoliquiritin, liquiritigenin, isoliquiritigenin, and dozens of compounds such as liquiritin, glycyl alcohol, isoliquiritigenin, 7-methyl coumarine and umbelliferone, and the Glabridin (Glabradin) is an internationally recognized liquorice whitening active ingredient.
Disclosure of Invention
The invention aims to provide a monomer component with high safety in liquorice and whitening and anti-aging activities.
In order to achieve the purpose, the invention adopts the following technical means:
according to the invention, main liquorice monomers (including liquiritigenin, isoliquiritigenin, isoliquiritin, Glabridin, glycyrrhizic acid and licochalcone A) are researched in the aspects of promoting collagen synthesis, inhibiting MMP1mRNA expression and toxicity to cells, and the like, and the result shows that although the Glabridin (Glabridin) is an internationally recognized liquorice whitening active ingredient, the Glabridin has toxicity to fibroblasts and can stimulate MMPs expression, so that certain potential safety hazards exist to a human body when the Glabridin is added into cosmetics as a whitening ingredient. Research proves that the other two substances, namely Isoliquiritigenin (Isoliquiritigenin) and isoliquiritin (Isoliquiritin) have obvious promotion effect on the type I collagen alpha 1 chain (Col1A1) and the Sirt1, can inhibit the expression of MMP1, are substances beneficial to improving the skin dermis, and have low cytotoxicity and high safety, so that the Isoliquiritigenin (Isoliquiritin) and the isoliquiritin (Isoliquiritin) have important application value in preparing the anti-aging cosmetic.
The molecular structural formulas of Isoliquiritigenin (Isooliquitinin) and isoliquiritin (Isooliquitinoside) are respectively shown as follows:
based on the research, the invention provides the application of isoliquiritigenin or isoliquiritin in preparing cosmetics with high safety and anti-aging effect. And
the isoliquiritigenin or isoliquiritin can be used for inhibiting apoptosis of human skin fibroblasts and promoting synthesis of collagen in human skin fibroblasts.
The isoliquiritigenin or isoliquiritin inhibits human skin fibroblast apoptosis by improving the expression level of the type I collagen alpha 1 chain (Col1A1) in human skin fibroblasts, reducing the expression level of matrix metalloproteinase 1(MMP1) and promoting the expression level of silent information regulator 1(Sirt1), promotes the synthesis of collagen in human skin fibroblasts and further achieves the anti-aging purpose.
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FIG. 1 is a graph of the effect of licorice monomer on SIRT1 activity.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
Example 1 cytotoxicity of Glycyrrhiza monomer
In order to determine the cytotoxicity of each licorice monomer in the licorice fermentation broth, the invention tests the cytotoxicity of 6 licorice monomers on human skin fibroblasts (nHDFs) by the WST-1 method, and the test results are shown in Table 1.
TABLE 1 cytotoxicity of Glycyrrhiza active monomers on fibroblasts at different concentrations
Example 2 Effect of Glycyrrhiza monomers on the expression of COLLA1, MMP1mRNA
1. Instruments and reagents
2. Test method
The changes in the amounts of mRNA of the representative genes of type 1 collagen and collagenase that degrades type 1 collagen were measured by a real-time fluorescent quantitative PCR (qRT-PCR) assay to determine the changes in type 1 collagen and collagenase. Human skin fibroblast (nHDF) cells were plated at 3X10 in 60mm plates5After one well, 24 hours of culture was performed. Then, a sample solution of a certain concentration was added thereto and the incubation was further carried out for 24 hours. After harvesting the cultured cells, 1ml of TRIzol reagent (Life Technology) was added to lyse the cells and extract their total mRNA. By using
The concentration and purity of the total mRNA are tested by a ultramicro spectrophotometer, (wherein only the total mRNA with the purity of more than 2.0 is selected). Total mRNA was reverse transcribed into cDNA using reverse transcriptase (M-MLV reverse transcriptase) and used for qRT-PCR. qRT-PCR was performed using HOT FIREPol EvaGreen PCR Mix Plus, and the expression of the gene was analyzed using the StepOnePelus RT-PCR system. The PCR was carried out under conditions of denaturation at 94 ℃ for 5 minutes, denaturation at 94 ℃ for 30 seconds, annealing at 60 ℃ for 30 seconds, and elongation at 72 ℃ for 30 seconds for a total of 40 cycles. Gene tableThe change in expression was determined by comparing the expression levels of the β -ACTIN control gene.
2. Test results
Evaluation of the effect of glycyrrhiza monomer on nHDF cells Col1a1 and MMP1 was performed by measuring the expression amount of mRNA of Col1a1 and MMP 1. The results are shown in Table 2.
TABLE 212 h Effect of Glycyrrhiza monomers on COL1A1 and MMP1mRNA
The above experiments were conducted to investigate the effect of isoliquiritigenin and isoliquiritin on the expression of mRNA of Col1a1 and MMP1 at different concentrations, and the data results are shown in table 3:
TABLE 324 h Effect of Glycyrrhiza monomers on COL1A1 and MMP1mRNA
From the results of comprehensive experiments, it can be judged that isoliquiritigenin, isoliquiritin and licochalcone extracted from liquorice have the effect of increasing Col1A 1; isoliquiritigenin and isoliquiritin can reduce MMP1 content. Therefore, isoliquiritigenin and isoliquiritin can increase Col1A1 and reduce the expression of MMP1, and are substances which are helpful for improving the dermis layer of the skin. Glabridin can reduce COL1A1 content and increase MMP-1 expression. And has potential safety hazard due to high cytotoxicity. The glabrene and the glabroside have obvious promotion effect on Col1A1 at lower concentration, can inhibit the expression of MMP1, have low cytotoxicity and high safety, and have important application value in cosmetics for promoting collagen synthesis and resisting aging.
Example 3 Effect of Glycyrrhiza monomers on SIRT1 Activity
1. Instruments and reagents
2. Test method
In order to determine the application prospect of the liquorice active monomer in the anti-aging field, the activity of the non-aging gene (SIRT1) is promoted by each monomer.
Silent information regulator type 1(Sirt1) is NAD+Dependent deacetylases, colloquially referred to as the immortal genes, were the first discovered members of the Sirtuin family of proteins in mammals. SIRT1 is a key molecule for delaying senility and prolonging life of mammals, can interact with chromatin, a plurality of important transcription factors and transcription co-regulatory factors, regulates gene transcription, chromosome stability and target protein activity through deacetylation, and plays a wide regulation role in cell cycle, metabolism, differentiation and the like. Research shows that the high expression of SIRT1 can inhibit apoptosis and promote the expression of extracellular matrix genes and collagen.
The activity of SIRT1 deacetylase was determined using the SIT1 kit according to the methods described in the instructions for use. This process is carried out by a two-step enzymatic reaction. In the first step, SIRT1 reacts at the acetylated lysine side chain of SIRT1 substrate, acting during deacetylation. And secondly, according to the developer, a fluorophore is obtained by cutting the deacetylated substrate, and the obtained fluorescence is used for measuring the activity of SIRT1 by using a fluorescence photometer.
The specific experimental method comprises the following steps: resveratrol (SIR1 agonist as positive control), nicotinamide (SIRT1 inhibitor as negative control), liquiritigenin, isoliquiritigenin, isoliquiritin, glabridin, glycyrrhizic acid, licochalcone A with different concentrations were added into the cell culture medium, respectively, and cell matrix was taken and reacted at 37 deg.C for 30 min. At this time, the purified SIRT1 enzyme was brought as close to 1.5ug as possible by a sufficient reaction. After adding a developer and mixing them thoroughly, the mixture was reacted at 37 ℃ for 10 minutes, and then the absorbance at 366nm or 435nm was measured by a microplate reader.
3. Statistical analysis method
The statistical treatment of this experiment was analyzed using Excel. For the purpose of analyzing the experimental results, the average value, standard deviation, percentage, etc. were used in the repeated experimental results; to analyze whether the results of the machine measurements for scavenging cytotoxicity and reactive oxygen species were meaningful changes, student's t-test analysis (conditions: bilateral analysis, equi-variance) was used.
4. Test results
The expression and activity of SIRT1 in cells are finely regulated, and the influence of isoliquiritigenin and isoliquiritin and other four major monomers of licorice on the regulation change of SIRT1 activity is studied by using a method for measuring the activity of SIRT1 in vitro, and the result is shown in figure 1.
The research shows that the isoliquiritigenin and the isoliquiritin can realize the high expression of SIRT1, so that the isoliquiritigenin and the isoliquiritin have the effects of inhibiting apoptosis and promoting the expression of extracellular matrix genes and collagen.
Claims (3)
1. The application of the isoliquiritin in preparing the cosmetics with high safety and anti-aging effect is characterized in that the isoliquiritin has the function of promoting the synthesis of collagen of human skin fibroblasts, and the concentration of the isoliquiritin is 20 mu M.
2. The use according to claim 1, wherein the isoliquiritin achieves the anti-aging purpose by increasing the expression level of the type i collagen alpha 1 chain (Col1a1) in human skin fibroblasts, reducing the expression level of matrix metalloproteinase 1(MMP1), and promoting the expression level of sirtuin 1(Sirt 1).
3. The use according to claim 1, wherein the isoliquiritin is used for inhibiting apoptosis of human skin fibroblasts and promoting synthesis of collagen in human skin fibroblasts by increasing the expression level of type i collagen alpha 1 chain (ColA 1), reducing the expression level of matrix metalloproteinase 1(MMP1), and promoting the expression level of sirtuin 1(Sirt 1).
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