Cosmetic containing silk tree bark extract and thioredoxin

Abstract

The present invention addresses the problem of providing: a cosmetic in which the elastin expression-promoting effect of thioredoxin is enhanced; and a novel elastin expression promoter. As a solution for the problem, the present invention provides: a cosmetic containing a silk tree bark extract and thioredoxin; and a dermal elastin expression promoter containing a silk tree bark extract.

Description

Cosmetics containing albizia julibrissin bark extract and thioredoxin

[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.

[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 this led to the present invention.

[0003] Japanese Patent Application Laid-Open No. 2016-216436

[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.

[0005] The main components of the present invention are as follows: 1. A cosmetic containing an extract of Albizia julibrissin bark and thioredoxin. 2. A promoter for promoting dermal elastin expression containing an extract of Albizia julibrissin bark.

[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.

[0007] Figure 1 is a graph showing the elastin expression promoting effect of Albizia julibrissin bark extract, Figure 2 is a graph showing the elastin expression promoting effect of thioredoxin, and Figure 3 is a graph showing the elastin expression promoting effect of a combination of Albizia julibrissin bark extract and thioredoxin.

[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. Dried silk tree bark is a traditional Chinese medicine called "hemanpi," and is used as a tonic, stimulant, sedative, analgesic, anthelmintic, and diuretic. Silk tree bark extract is preferably extracted by heating with an aqueous solvent. Examples of aqueous solvents include water, ethanol, propylene glycol, dipropylene glycol, and 1,3-butanediol. Silk tree bark extract can be commercially available, such as Silk Tree Bark Extract (cosmetic labeling name) manufactured by Toyobo Co., Ltd. The amount of silk tree bark extract blended into cosmetics 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 discovered in Escherichia coli in 1964 as a coenzyme for ribonucleotide reductase, essential for DNA synthesis, and is ubiquitous in all living organisms. Thioredoxin may be a commercially available purified product used as a reagent, or it may be a human recombinant protein. Thioredoxin, a human recombinant protein, is a recombinant peptide produced using E. coli called human oligopeptide-4, and a commercially available product such as "recombinant human thioredoxin" manufactured by Oriental Yeast Co., Ltd. The amount of thioredoxin to be incorporated into cosmetics 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 blending 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.

[0012] [Effect of Albizia julibrissin bark extract and thioredoxin on promoting elastin expression] 1. Sample preparation: 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%. The 2% FBS-containing DMEM solution was used as an unsupplemented sample. Albizia julibrissin bark extract manufactured by Toyobo Co., Ltd. was used. 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. Thioredoxin, human, recombinant [rADF], a human recombinant thioredoxin protein manufactured by Oriental Yeast Co., Ltd., was used.

[0013] 2. Cell culture and total RNA extraction Normal human dermal fibroblasts (Neo) (hereafter referred to as NHDF) (Thermo Fisher Scientific) were cultured in 10% FBS-containing DMEM (Thermo Fisher Scientific) with the addition of penicillin (Fujifilm Wako Pure Chemical Industries). When the cells reached 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), resuspended in culture medium, and then cultured at 5 x 10 4 500 μl of each sample was seeded into a 24-well plate at 100 cells / well. After culturing for 1 day in a 37°C, 5% CO2 incubator, 500 μl of a sample prepared with culture medium was added to each well. 24 hours later, the culture supernatant was collected. After the supernatant was collected, the cells were washed once with PBS(-) and RNA iso (TAKARA) was added to each well at 200 μl / well to extract total RNA.

[0014] 3. Total RNA Extraction Method: ・200 μl of RNAiso was added per well, and RNA was extracted using the following steps. ・The cells were pipetted at least 10 times and collected in a 1.5 ml centrifuge tube. ・The tube was left to stand at room temperature (15-30°C) for 5 minutes to release nuclear proteins from nucleic acids. ・50 μl of chloroform was added, and the tube was shaken until the mixture became milky white, and then left to stand at room temperature for 10 minutes. ・The tube was centrifuged at 12,000×g for 15 minutes at 4°C, and the upper aqueous layer was transferred to a new centrifuge tube. ・100 μl of isopropanol was added, mixed well, and left to stand at room temperature for 10 minutes. ・The tube was centrifuged at 12,000×g for 15 minutes at 4°C to precipitate RNA. Discard the supernatant, add 200 μl of 75% cold ethanol to wash the precipitate, and centrifuge at 7,500 × g for 5 minutes at 4°C. Discard the supernatant, add 15 μl of RNase-free water, and dissolve the precipitate (RNA).

[0015] 4. PCR: Total RNA was quantified using a NanoDrop 2000C (Thermo Scientific) and the concentration was adjusted. Reverse transcription was performed according to the protocol of the PrimeScript® RT reagent Kit (TAKARA). The RNA was prepared using SYBR® Premix Ex Taq® II (Tli RNaseH Plμs) (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 for 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. 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 by 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. The human recombinant thioredoxin protein promoted elastin (ELN) expression in skin fibroblasts at concentrations of 0.0003% to 0.003% (Figure 2).

[0018] [Effect of a combination of Albizia julibrissin bark extract and thioredoxin on promoting elastin expression] 1. Preparation of combination samples Samples with the following concentrations were prepared in a 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 served as an unsupplemented sample. Albizia julibrissin bark extract manufactured by Toyobo Co., Ltd. was used. 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. Thioredoxin, human, recombinant [rADF], a human recombinant thioredoxin protein manufactured by Oriental Yeast Co., Ltd., was used.

[0019] 2. Cell culture and total RNA extraction were 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 was performed in the same manner as in the test "Effect of Albizia julibrissin bark extract and thioredoxin on promoting elastin expression."

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

[0022] 5. Elastin Expression Promoting Effect of a Combination of Albizia Julibrissin Bark Extract and Thioredoxin Figure 3 shows the results for the addition of Albizia Julibrissin bark extract alone, thioredoxin alone, and both Albizia Julibrissin bark extract and thioredoxin. The amount of elastin expressed in skin fibroblasts was 1.6-fold with the addition of 0.001% thioredoxin and 1.4-fold with the addition of 0.0000025% Albizia Julibrissin bark extract compared to the control. The theoretical value for 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-fold compared to the control, but the actual value measured when 0.001% thioredoxin and 0.0000025% Albizia Julibrissin bark extract were added simultaneously was 2.3-fold. The amount of elastin expressed in skin fibroblasts was 1.4 times higher when 0.00001% Albizia julibrissin bark 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 bark extract were added simultaneously should be 2.0 times higher than the control, but the actual value measured when 0.001% thioredoxin and 0.00001% Albizia julibrissin bark extract were added simultaneously was 2.9 times higher. A greater effect was confirmed by combining Albizia julibrissin bark extract and thioredoxin.

Claims

1. A cosmetic product containing albizia julibrissin bark extract and thioredoxin.

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

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