Cosmetics containing albizia julibrissin bark extract and thioredoxin

Abstract

An object of the present invention is to provide a cosmetic preparation in which the elastin expression promoting effect of thioredoxin is enhanced, and to provide a new elastin expression promoting agent. The present invention provides a cosmetic containing an extract of Albizia julibrissin bark and thioredoxin, and a dermal elastin expression promoter containing an extract of Albizia julibrissin bark.

Description

[Technical Field]

[0001] The present invention relates to a cosmetic preparation containing an extract from Albizia julibrissin bark and thioredoxin, and to a dermal elastin expression promoter containing an extract from Albizia julibrissin bark. [Background technology]

[0002] Elastin is involved in skin elasticity, and there is a need for topical agents that promote the expression of elastin. Thioredoxin is known as an elastin expression promoter (Patent Document 1). In order to search for a material that can enhance the effects of thioredoxin, the present inventor evaluated an extract of Albizia julibrissin bark, which is known as an antioxidant, and arrived at the present invention. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Laid-Open No. 2016-216436 Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide a cosmetic preparation in which the elastin expression promoting effect of thioredoxin is enhanced, and to provide a new elastin expression promoting agent. [Means for solving the problem]

[0005] The main configuration of the present invention is as follows. 1. A cosmetic product containing albizia julibrissin bark extract and thioredoxin. 2. A dermal elastin expression promoter containing albizia julibrissin bark extract. [Effects of the Invention]

[0006] By blending Albizia julibrissin bark extract and thioredoxin into cosmetics, we have been able to provide cosmetics that effectively promote the expression of elastin.We have also been able to provide an elastin expression promoter containing Albizia julibrissin bark extract. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 is a graph showing the elastin expression promoting effect of an extract from Albizia julibrissin bark. [Figure 2] FIG. 2 is a graph showing the effect of thioredoxin in promoting elastin expression. [Figure 3] FIG. 3 is a graph showing the effect of promoting elastin expression by a combination of Albizia julibrissin bark extract and thioredoxin. DETAILED DESCRIPTION OF THE INVENTION

[0008] The cosmetic of the present invention contains an extract of Albizia julibrissin bark and thioredoxin.

[0009] Silk tree is a legume plant, and its scientific name is Albizzia julibrissin Durazz. The dried bark of silk tree is used as a traditional Chinese medicine called "Hehuanpi" and is used as a tonic, stimulant, sedative, analgesic, anthelmintic, and diuretic. The Albizia julibrissin bark extract is preferably extracted by heating with an aqueous solvent, such as water, ethanol, propylene glycol, dipropylene glycol, or 1,3-butanediol. As the Albizia julibrissin bark extract, commercially available products can be used, for example, Albizia julibrissin bark extract (labeled name for cosmetics) manufactured by Toyobo Co., Ltd. The amount of Albizia julibrissin bark extract blended into the cosmetic is preferably 0.0000001% by mass or more and 0.0005% by mass or less, and particularly preferably 0.0000005% by mass or more and 0.00025% by mass or less.

[0010] Thioredoxin is a protein thiol-disulfide oxidoreductase that was discovered in Escherichia coli in 1964 as a coenzyme of ribonucleotide reductase, which is essential for DNA synthesis, and is found universally in all living organisms. Thioredoxin can be a commercially available purified product used as a reagent, or it can be a human recombinant protein. Human recombinant thioredoxin is a recombinant peptide produced using E. coli called human oligopeptide-4, and a commercially available product that can be used is "recombinant human thioredoxin" manufactured by Oriental Yeast Co., Ltd. The amount of thioredoxin blended into the cosmetic is preferably 0.00005% by mass or more and 0.005% by mass or less, and particularly preferably 0.0003% by mass or more and 0.003% by mass or less. The blend ratio of the albizia julibrissin bark extract to thioredoxin is preferably in the range of from albizia julibrissin:thioredoxin=1:5000 to albizia julibrissin:thioredoxin=1:0.1, and particularly preferably in the range of from albizia julibrissin:thioredoxin=1:5000 to albizia julibrissin:thioredoxin=1:10.

[0011] The cosmetic of the present invention can be provided in various dosage forms such as lotions, emulsions, gels, creams, ointments, etc. Furthermore, various additives for formulation can be blended as needed. [Example]

[0012] [Effect of Albizia julibrissin bark extract and thioredoxin on promoting elastin expression] 1. Preparation of a Single Sample Samples with the following concentrations were prepared in 2% FBS-containing DMEM solution: Albizia julibrissin bark extract 0.0000005%, 0.0000025%, 0.00001%, 0.00005%, 0.00025%, and thioredoxin 0.0003%, 0.001%, and 0.003%. 2% FBS-containing DMEM solution was used as a non-additive sample. The Albizia julibrissin bark extract used was manufactured by Toyobo Co., Ltd. The Albizia julibrissin bark extract manufactured by Toyobo Co., Ltd. is provided as a solution, and the concentration of the Albizia julibrissin bark extract was determined by the amount of extract added to the solution. The thioredoxin used was human recombinant thioredoxin protein, Thioredoxin, Hμman, recombinant [rADF] manufactured by Oriental Yeast Co., Ltd.

[0013] 2. Cell Culture and Total RNA Extraction Normal human dermal fibroblasts (Neo) (NHDF) (Thermo Fisher Scientific) were cultured in 10% FBS-containing DMEM (Thermo Fisher Scientific) supplemented with penicillin (Fujifilm Wako Pure Chemical Industries). When the cells reached approximately 80-90% confluence, they were detached with 0.025% trypsin / 0.01% EDTA (0.05% trypsin / 0.02% EDTA, Sigma, diluted 2-fold with PBS). After resuspending in culture medium, 500 μl of the cells were seeded into a 24-well plate at 5 x 104 cells / well. After 1 day of culture in a 37°C, 5% CO2 incubator, 500 μl of the sample prepared in culture medium was added to each well. The culture supernatant was then collected 24 hours later. After collecting the supernatant, the cells were washed once with PBS(-), and 200 μl / well of RNA iso (TAKARA) was added to extract total RNA.

[0014] 3.Total RNA extraction method RNAiso was added at 200 μl / well, and RNA was extracted using the following steps. The cells were pipetted more than 10 times and collected in a 1.5 ml centrifuge tube. Leave at room temperature (15-30°C) for 5 minutes to release nucleoproteins from nucleic acids. Add 50 μl of chloroform, shake in a centrifuge tube until the mixture becomes milky white, and let stand at room temperature for 10 minutes. Centrifuge at 12,000 x g for 15 minutes at 4°C and transfer the upper aqueous layer to a new centrifuge tube. Add 100 μl of isopropanol, mix well, and let stand at room temperature for 10 minutes. Centrifuge at 12,000 x g for 15 minutes at 4°C to precipitate the RNA. Discard the supernatant, add 200 μl of cold 75% ethanol to wash the pellet, and centrifuge at 7,500 × g for 5 minutes at 4°C. Discard the supernatant and add 15 μl of RNase-free water to dissolve the precipitate (RNA).

[0015] 4. PCR method Total RNA was quantified using a NanoDrop 2000C (Thermo Scientific) and adjusted for concentration. Reverse transcription was performed according to the protocol of the PrimeScript® RT reagent Kit (TAKARA). RNA was prepared using SYBR® Premix Ex Taq® II (Tli RNaseH P1μs) (TAKARA) and then subjected to real-time PCR using a LightCycler® 480 (Roche). The genes used were RPS18 (GenBank NM_080704.4), gene encoding protein ribosomal protein S18, primer name Homo sapiens ribosomal protein S18 (RPS18), mRNA, manufactured by TAKARA, and ELN (GenBank NM_000501.3), gene encoding protein elastin, primer name Homo sapiens elastin (ELN), transcript variant 1, mRNA, manufactured by TAKARA. After completion, the ΔCp value for RPS18 was calculated, and the ΔΔCp value for the average value of RPS18 in the control was calculated, and a graph was created using 2ΔΔCp.

[0016] 5. Data Analysis N3 samples were collected for each condition, and the ratio to the untreated sample was calculated and a significant difference test (t-test) was performed.

[0017] 6. The promoting effect of thioredoxin and Albizia julibrissin bark extract on elastin expression Albizia julibrissin bark extract promoted elastin expression in skin fibroblasts at concentrations of 0.0000005% to 0.00025% (Figure 1). Compared to the 0% control, the addition of 0.0000005% Albizia julibrissin bark extract promoted elastin expression 1.24-fold, 1.35-fold at 0.0000025%, 1.38-fold at 0.00001%, 1.68-fold at 0.00005%, and 1.50-fold at 0.00025%. Albizia julibrissin bark extract is effective as an agent for promoting dermal elastin (ELN) expression. Human recombinant thioredoxin protein promoted elastin (ELN) expression in skin fibroblasts at concentrations of 0.0003% to 0.003% (Figure 2).

[0018] [Effect of the combination of Albizia julibrissin bark extract and thioredoxin on promoting elastin expression] 1. Combination Sample Preparation Samples with the following concentrations were prepared in 2% FBS-containing DMEM solution: Albizia julibrissin bark extract 0.0000025%, 0.00001%, thioredoxin 0.001%, (Albizia julibrissin bark extract and thioredoxin) (0.0000025% and 0.001%), (0.00001% and 0.001%). The 2% FBS-containing DMEM solution was used as an unsupplemented sample. The Albizia julibrissin bark extract used was manufactured by Toyobo Co., Ltd. The Albizia julibrissin bark extract manufactured by Toyobo Co., Ltd. is provided as a solution, and the concentration of the Albizia julibrissin bark extract was determined by the amount of extract added to the solution. The thioredoxin used was human recombinant thioredoxin protein, Thioredoxin, Hμman, recombinant [rADF] manufactured by Oriental Yeast Co., Ltd.

[0019] 2. Cell Culture and Total RNA Extraction The test was carried out in the same manner as in the test "Effect of Albizia julibrissin bark extract and thioredoxin on promoting elastin expression."

[0020] 3.Total RNA extraction method The test was carried out in the same manner as in the test "Effect of Albizia julibrissin bark extract and thioredoxin on promoting elastin expression."

[0021] 4. PCR method The test was carried out in the same manner as in the test "Effect of Albizia julibrissin bark extract and thioredoxin on promoting elastin expression."

[0022] 5. The combination of Albizia julibrissin bark extract and thioredoxin promotes elastin expression FIG. 3 shows the results when the Albizia julibrissin bark extract alone was added, when thioredoxin alone was added, and when the Albizia julibrissin bark extract and thioredoxin were added. The amount of elastin expressed in skin fibroblasts was 1.6 times higher with the addition of 0.001% thioredoxin and 1.4 times higher with the addition of 0.0000025% Albizia julibrissin bark extract compared to the control. The theoretical value of the amount of elastin expressed in skin fibroblasts when 0.001% thioredoxin and 0.0000025% Albizia julibrissin bark extract were added simultaneously should be 2.0 times higher than the control, but the actual value measured when 0.001% thioredoxin and 0.0000025% Albizia julibrissin bark extract were added simultaneously was 2.3 times higher. The amount of elastin expressed in skin fibroblasts was 1.4 times higher when 0.00001% Albizia julibrissin extract was added compared to the control. The theoretical value of the amount of elastin expressed in skin fibroblasts when 0.001% thioredoxin and 0.00001% Albizia julibrissin extract were added simultaneously should be 2.0 times higher than the control, but the actual value measured when 0.001% thioredoxin and 0.00001% Albizia julibrissin extract were added simultaneously was 2.9 times higher. A combination of Albizia julibrissin bark extract and thioredoxime was found to be even more effective.

Claims

1. A cosmetic containing an extract of Albizia julibrissin (silk tree) bark and thioredoxin, wherein the blending amount of the extract of Albizia julibrissin (silk tree) bark is 0.0000005% by mass or more and 0.00025% by mass or less, and the blending amount of the thioredoxin is 0.0003% by mass or more and 0.003% by mass or less.

2. A dermal elastin expression promoter containing albizia julibrissin bark extract and thioredoxin.

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