Anti-aging composition as well as preparation method and application thereof

Abstract

The invention belongs to the field of cosmetics, and discloses an anti-aging composition as well as a preparation method and application thereof. The anti-aging composition is prepared from the following components in percentage by mass: 0.01 percent to 0.2 percent of a mutant sr-(tripeptide-133 sh-polypeptide-2 hexapeptide-40), 0.1 percent to 1.0 percent of glycosylglycerol, 0.01 percent to 0.15 percent of hydroxyl pinacolone retinoate, 0.05 percent to 0.5 percent of ceramide NP, 0.05 percent to 0.5 percent of ceramide AP, 0.1 percent to 0.5 percent of resveratrol and 97.15 percent to 99.68 percent of a basic component, wherein the basic components comprise water, a thickening agent, a preservative and a penetration enhancer. The mutant sr-(tripeptide-133sh-polypeptide-2 hexapeptide-40) with high antioxidant activity is obtained by carrying out double-site combination site-directed mutagenesis on wild thioredoxin, and the mutant sr-(tripeptide-133sh-polypeptide-2 hexapeptide-40) and various active ingredients are synergistically acted to develop a brand-new efficient active raw material combination which can be applied to skin care and washing care products and can improve the effects of oxidation resistance, repair and the like of the products.

Description

Technical Field

[0001] This application belongs to the field of cosmetics, and in particular relates to an anti-aging composition, its preparation method and application. Background Technology

[0002] Aging is a degenerative physiological process characterized by a decline in biological function and a reduction in adaptability and resistance. Oxidative stress damage is closely related to aging. Under normal circumstances, the body maintains a dynamic balance between oxidation and antioxidation. However, with increasing age, an excess of free radicals and / or a deficiency of antioxidants lead to the destruction of biological membrane structures and the impairment of organelle functions, ultimately disrupting this dynamic balance. In the aerobic metabolism of aging, increased free radicals or a lack of antioxidants increase cytotoxicity and reduce the body's antioxidant capacity, resulting in irreversible damage to biological membranes and DNA molecular structures, leading to dry skin, loss of elasticity, and wrinkles.

[0003] Thioredoxin (Trx) is a widely distributed, heat-stable protein that acts as a hydrogen carrier. Natural thioredoxin (Trx), as an important redox regulator, can scavenge free radicals and regulate cellular redox status. However, its wild-type form suffers from low expression levels and insufficient antioxidant activity, limiting its application in skincare products. While research on genetically modifying thioredoxin has been conducted, studies on its antioxidant and cell repair aspects are limited. Currently, the demand for anti-aging products is increasing rapidly. Our company has developed a novel thioredoxin mutant and, based on this mutant, has developed an anti-aging composition. Summary of the Invention

[0004] The purpose of this application is to provide an anti-aging composition, its preparation method and application, and in particular, to provide an anti-aging composition based on a thioredoxin mutant, its preparation method and application.

[0005] This application discloses an anti-aging composition, which, by mass percentage, comprises mutant sr-(tripeptide-133 sh-polypeptide-2 hexapeptide-40) (0.01%-0.2%), glyceryl glucoside (0.1-1.0%), hydroxypinazone retinate (0.01-0.15%), ceramide NP (0.05-0.5%), ceramide AP (0.05-0.5%), resveratrol (0.1-0.5%), and a base component (97.15%-99.68%); wherein the base component includes water, thickener, preservative, and penetration enhancer.

[0006] Preferably, the base components include 0.5-3% preservative, 0.1-1.5% thickener, 10-40% penetration enhancer, and water to make up the balance.

[0007] Preferably, the content of the basic component is 98.45%-98.84% by mass percentage, more preferably 98.65%-98.84%, and most preferably 98.65%-98.8%.

[0008] Preferably, the anti-aging composition, by mass percentage, comprises mutant sr-(tripeptide-133 sh-polypeptide-2 hexapeptide-40) (0.05%-0.2%), glyceryl glucoside (0.5%), hydroxypinazone retinate (0.05%), ceramide NP (0.2%), ceramide AP (0.1%), resveratrol (0.3%), and a base component of 98.65%-98.8%; wherein the base component comprises preservative 2%, thickener 0.6%, penetration enhancer 30.6%-31.6%, and water to make up the balance.

[0009] Preferably, the preservative is selected from one of 1,2-hexanediol, pentanediol, p-hydroxyacetophenone and phenoxyethanol, and is preferably 1,2-hexanediol.

[0010] Preferably, the thickener is selected from one or more of xanthan gum, acrylamide dimethyl taurate ammonium / VP copolymer, carbomer, and hydroxyethyl cellulose. Preferably, it is a composition of xanthan gum and acrylamide dimethyl taurate ammonium / VP copolymer, with a mass percentage ratio of xanthan gum: acrylamide dimethyl taurate ammonium / VP copolymer of 1:1 to 1:10, for example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, preferably 1:5.

[0011] Preferably, the penetration enhancer is one or more of hydrogenated lecithin, 2,3-butanediol, laurocapram, 1,3-butanediol, 1,3-propanediol, and polysorbate. More preferably, the penetration enhancer comprises hydrogenated lecithin, 2,3-butanediol, and laurocapram, wherein, by mass percentage, hydrogenated lecithin accounts for 0.1-1.5% of the total mass of the composition; 2,3-butanediol accounts for 30% of the total mass of the composition; and laurocapram accounts for 0.1% of the total mass of the composition. More preferably, by mass percentage, hydrogenated lecithin accounts for 0.5-1.5% of the total mass of the composition; 2,3-butanediol accounts for 30% of the total mass of the composition; and laurocapram accounts for 0.1% of the total mass of the composition.

[0012] Preferably, the anti-aging composition, by mass percentage, comprises 0.1% mutant sr-(tripeptide-133 sh-polypeptide-2 hexapeptide-40), 0.5% glyceryl glucoside, 0.05% hydroxypinazone retinate, 0.2% ceramide NP, 0.1% ceramide AP, 0.3% resveratrol, and 98.75% basic components; wherein the basic components include 2% 1,2-hexanediol, 0.1% xanthan gum, 0.5% acrylamide dimethyl taurate ammonium / VP copolymer, 1% hydrogenated lecithin, 30% 2,3-butanediol, 0.1% laurocapram, and water to make up the balance.

[0013] Preferably, the mutant is a thioredoxin mutant sr-(tripeptide-133 sh-polypeptide-2 hexapeptide-40), the amino acid sequence of which is shown in SEQ ID NO: 4. The gene encoding the thioredoxin mutant sr-(tripeptide-133 sh-polypeptide-2 hexapeptide-40) has the nucleotide sequence shown in SEQ ID NO: 5.

[0014] This application also discloses a method for preparing the above-mentioned anti-aging composition, comprising the following steps: Step 1: Mix the prescribed amounts of hydroxypinazone retinate, ceramide NP, ceramide AP, resveratrol, 2,3-butanediol, and laurocapram at 70-80℃ to obtain solution A; Step 2: Mix the prescribed amounts of hydrogenated lecithin, xanthan gum, acrylamide dimethyl taurate ammonium / VP copolymer, 1,2-hexanediol and water at 70-80℃ until homogeneous to obtain solution B; Step 3: Mix the prescribed amounts of mutant sr-(tripeptide-133 sh-polypeptide-2 hexapeptide-40) and glycerol glucoside at room temperature to obtain solution C; Step 4: Mix solutions A and B at 70-80℃ until homogeneous, homogenize using a homogenizer at 6000-7500 RPM for 3-5 minutes, cool to 40-45℃, add solution C, mix until homogeneous, and obtain the anti-aging composition.

[0015] This application also provides the use of the anti-aging composition in the preparation of skin care or personal care products.

[0016] As a technical solution, the skin care products can be serums, creams, lotions, and masks, etc.; the washing and care products can be facial cleansers, shower gels, and body lotions, etc.

[0017] This application also discloses an essence comprising the aforementioned anti-aging composition.

[0018] As one technical solution, the essence comprises the following components in the following mass percentages: 1,2-hexanediol 0.5-1.5%, phenoxyethanol 0.1-0.5%, glycerin 2-8%, hydroxyethyl cellulose 0.05-0.25%, sodium hyaluronate 0.1-0.3%, rose hydrosol 5-15%, anti-aging composition 0.5-5%, and the balance being water.

[0019] Preferably, the essence comprises the following components in the following weight percentages: 1,2-hexanediol 1%, phenoxyethanol 0.2%, glycerin 5%, hydroxyethyl cellulose 0.1%, sodium hyaluronate 0.2%, rose hydrosol 10%, anti-aging composition 2%, and the balance being water.

[0020] All percentages mentioned above refer to the mass percentage of each component relative to the total mass of the composition.

[0021] Beneficial effects: Compared with the prior art, this application has the following significant advantages: (1) This application obtains a mutant sr-(tripeptide-133sh-polypeptide-2 hexapeptide-40) with high antioxidant activity by performing a two-site combination site-directed mutagenesis on wild-type thioredoxin. Experiments show that the expression level of sr-(tripeptide-133sh-polypeptide-2 hexapeptide-40) in Escherichia coli is 120 mg / L and the soluble ratio is 100%; in the ABTS free radical scavenging experiment, the concentration in the range of 200~2000 μg / mL all showed significant antioxidant activity, with the highest scavenging rate reaching 52.6%, which is 27.4% higher than that of wild type. (2) The composition of this application integrates a variety of ingredients with different anti-aging mechanisms. The multiple ingredients work together to comprehensively combat skin aging and can be applied to skin care and washing products to enhance the antioxidant and repair effects of the products. It is a new and highly efficient combination of active raw materials. (3) The composition of this application utilizes a combination of penetration enhancers to deliver the complex active ingredients, which can significantly improve the transdermal absorption and permeability, solving the bottleneck problem that has always existed in the industry: the active ingredients cannot be well absorbed through the skin barrier. In addition, the selected combination of penetration enhancers is also a moisturizing ingredient in cosmetics. While promoting the absorption of active ingredients, it can also protect the structure of active ingredients and improve the skin feel, thus making the anti-aging effect better. Attached Figure Description

[0022] Figure 1 This is a protein SDS-PAGE gel image; Figure 2 The bar chart shows the ATBS radical scavenging rates of wild-type and sr-(tripeptide-133 sh-peptide-2 hexapeptide-40) at different concentrations. Figure 3 It is the cumulative transdermal permeability of SR-(tripeptide-133 sh-polypeptide-2 hexapeptide-40). Detailed Implementation

[0023] The present application will be described in detail below with reference to specific embodiments. The glycerol glucoside, hydroxypinazone retinate, ceramide NP, and ceramide AP in the embodiments were purchased from Nanjing Shengde Baitai Biotechnology Co., Ltd., Jiangsu Jianersheng Biotechnology Co., Ltd., Chongqing Zhihe Biopharmaceutical Co., Ltd., resveratrol, Royal DSM Group, hydrogenated lecithin, Lipoid Kosmetik AG, 2,3-butanediol, GS Caltex Corporation, laurocapram, Beijing Solarbio Technology Co., Ltd., xanthan gum, CPKelco, acrylamide dimethyl taurate ammonium / VP copolymer, Clariant, and 1,2-hexanediol were purchased from Osaka Organic Chemical Industry Co., Ltd.

[0024] Example 1: Construction and expression of the thioredoxin mutant sr-(tripeptide-133 sh-peptide-2 hexapeptide-40) (I) Construction of the thioredoxin mutant sr-(tripeptide-133 sh-polypeptide-2 hexapeptide-40) The wild-type human thioredoxin TXN sequence corresponds to UniProt P10599. This sequence is derived from the human gene TXN (NCBI gene ID: 7295) and the nucleic acid sequence was optimized based on the codons of E. coli.

[0025] The codon-optimized human thioredoxin TXN was synthesized by Genscript Biotech Inc. and constructed into the E. coli expression vector pET-28a (+) using seamless cloning, containing a C-terminal -6×His, with the target gene inserted into the NcoI and XhoI restriction sites.

[0026] The amino acid sequence of the wild-type human thioredoxin gene (TXN) is shown in SEQ ID NO.1, and the nucleic acid sequence is shown in SEQ ID NO.2. The amino acid sequence of the human thioredoxin mutant sr-(tripeptide-133sh-polypeptide-2 hexapeptide-40) is shown in SEQ ID NO.3, which is a mutation of D60N and C73S in the wild-type gene. The nucleotide sequence of the gene encoding the thioredoxin mutant sr-(tripeptide-133sh-polypeptide-2 hexapeptide-40) is shown in SEQ ID NO.4. Here, sr-(tripeptide-133sh-polypeptide-2 hexapeptide-40) is the INCI name of this thioredoxin mutant.

[0027] SEQ ID NO.1 (Wild-type TXN) MVKQIESKTAFQEALDAAGDKLVVVDFSATWCGPCKMIKPFFHSLSEKYSNVIFLEVDVDDCQDVASECEVKCMPTFQFFKKGQKVGEFSGANKEKLEATINELVHHHHHH SEQ ID NO.2 (Wild-type TXN) ATGGTGAAACAAATTGAAAGCAAAACCGCGTTTCAGGAGGCGCTGGATGCCGCGGGTGACAAATTAGTTGTGGTGGATTTTAGCGCGACCTGGTGCGGTCCGTGCAAAATGATTAAACCGTTTTTTCATAGCCTGAGCGAAAAATACAGCAATGTGATTTTCCTGGAAGTGGACGTGGATGATTGCCAGGATGTGGCGAGCGAATGCGAAGTGAAATGTATGCCGACCTTTCAGTTTTTCAAAAAAGGCCAGAAAGTGGGCGAATTTAGCGGTGCGAATAAAGAGAAACTGGAAGCGACCATTAACGAACTGGTTCATCATCACCACCATCATTAA SEQ ID NO.3 (Mutant sr-(Tripeptide-133 sh-Polypeptide-2 Hexapeptide-40)) MSKIKQIESKTAFQEALDAAGDKLVVVDFSATWCGPCKMIKPFFHSLSEKYSNVIFLEVDVNDCQDVASECEVKSMPTFQFFKKGQKVGEFSGANKEKLEATINELVHHHHHH SEQ ID NO.4 (Mutant sr-(Tripeptide-133 sh-Polypeptide-2 Hexapeptide-40)) ATGTCTAAAATAAAACAAATTGAAAGCAAAACCGCGTTTCAGGAGGCTGGATGCCGCGGGTGACAAATTAGTTGTGGTGGATTTTAGCGCGACCTGGTGCGGTCCGTGCAAAATGATTAAACCGTTTTTTCATAGCCTGAGCGAAAAATACAGCAATGTGATTTTCCTG GAAGTGGACGTGAACGATTGCCAGGATGTGGCGAGCGAATGCGAAGTGAAAAGCATGCCGACCTTTCAGTTTTTCAAAAAAGGCCAGAAAGTGGGCGAATTTAGCGGTGCGAATAAAGAGAAACTGGAAGCGACCATTAACGAACTGGTTCATCATCACCACCATCATTAA Using the wild-type thioredoxin gene (UniProt P10599) as a template, site-directed mutagenesis primers were designed to introduce D60N, C73S, D60N / C73S double-site mutations, and D60N / C62S / C69S / C73S quad-site mutations into the P10599 gene. The primer sequences are shown in Table 1. PCR amplification was used to obtain the PCR product of the thioredoxin mutant. The reaction system consisted of 25 μL of 2 × Phanta Max Master Mix (P515-01, Vazyme), 2 μL (10 μM) of upstream primer, 2 μL (10 μM) of downstream primer, 1 μL (50 ng) of plasmid template, and ddH2O added to a final volume of 50 μL.

[0028] Table 1 Primer sequences

[0029] PCR amplification conditions: (1) Pre-denaturation: 95℃ for 3 min; (2) Denaturation: 95℃ for 15 s, Annealing: annealing for 15 s with the average Tm of the primers used, Extension: 72℃ for 30~60 sec / kb, for a total of 30 cycles; (3) Post-extension: 72℃ for 10 min; (4) Storage at 4℃. After amplification, the DNA template in the product was removed using SwiftCut Dpn Ⅰ (C404-01, Vazyme), and then recombined using ClonExpress Ultra One Step Cloning Kit (C115-01, Vazyme), and transformed into DH5α competent cells (C502-02, Vazyme) to circularize the PCR product. The transformed competent cell culture was plated on LB solid medium containing 50 μg / mL kanamycin (Kan) and cultured at 37°C for 15 h. Single clones were selected and transferred to LB test tubes (5 mL LB liquid medium containing 50 μg / mL kanamycin (Kan)) and cultured on a shaker at 37°C for 8-10 h. Then, thioredoxin and its mutant plasmids were extracted using the FastPure Plasmid Mini Kit (DC201-01, Vazyme). The mutant plasmids with correct sequences were confirmed by DNA sequencing.

[0030] (ii) Recombinant expression of thioredoxin mutants Preparation of recombinant genetically engineered bacteria: The obtained recombinant vector and its mutant plasmid were transformed into BL21(DE3) competent cells (C504-02, Vazyme), respectively, and plated on LB agar plates containing 50 μg / mL Kan. The plates were then incubated overnight at 37°C. Single colonies were picked from the plates and inoculated into LB test tubes, cultured with shaking at 37°C for 8-12 hours, and the bacterial strains were stored at -80°C. These are the mutant recombinant genetically engineered bacteria.

[0031] Culture of bacteria: Recombinant genetically engineered bacteria containing the coding sequences of wild-type thioredoxin and its mutants were inoculated into LB liquid medium containing Kan resistance (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and deionized water as solvent). Test tubes containing 4 mL of LB liquid medium (containing a final concentration of 50 μg / mL Kan) were placed in a shaker at 37°C and cultured at 200 rpm for 10-12 h to obtain seed culture.

[0032] Protein induction expression: The seed culture was transferred to a shake flask containing 1 L LB liquid medium (with a final concentration of 50 μg / mL Kan) in a clean bench and incubated at 37°C and 200 rpm for 2-3 hours. The OD of the culture medium was then measured. 600When the pH value reached 0.6-0.8, 0.2 mM IPTG was added to induce expression at a final concentration of 17℃. After 15 h of induction, the cells were collected by centrifugation and resuspended in a lysis buffer (50 mM Tris-HCl pH=7.4, 150 mM NaCl, 20 mM imidazole, 10% (v / v) glycerol, 0.1% (v / v) Tween 20). Benzyl sulfonyl fluoride (PMSF, final concentration 1 mM) was added before lysis.

[0033] Protein purification: E. coli cells were lysed using a pre-cooled high-pressure homogenizer, centrifuged at 9000 rpm for 45 min, and the supernatant was collected. The supernatant and Ni²⁺-NET medium were mixed by rotation at 4 °C for 1 h to allow the protein with 6×His to bind to the medium. The binding buffer was added to a gravity column and allowed to flow out naturally under gravity. The column was washed with 10 times its volume of lysis buffer, and then the eluent was collected into a 30 kDa ultrafiltration tube using an elution buffer (50 mM Tris-HCl pH=7.4, 150 mM NaCl, 300 mM imidazole, 10% (v / v) glycerol, 0.1% (v / v) Tween 20, 20 mM β-mercaptoethanol). The protein was concentrated by centrifugation and the replacement buffer was used. The protein was stored in a replacement buffer (50 mM Tris-HCl pH=7.4, 150 mM NaCl, 10% (v / v) glycerol, 0.1% (v / v) Tween 20). The protein concentration was determined using a BCA protein quantification kit, and the expression of the target protein was detected by protein electrophoresis.

[0034] Protein expression is shown in Table 2 and Figure 1 As shown, Figure 1 In the electrophoresis diagram, lane 5 contained a 180 kDa Prestained Protein Marker (MP102-01, Vazyme) with a loading volume of 5 μL; lanes 1 and 2 contained the inclusion body and soluble components of wild-type TXN in the lysis buffer, respectively; lanes 3 and 4 contained the inclusion body and soluble components of the mutant sr-(tripeptide-133 sh-peptide-2 hexapeptide-40) in the lysis buffer, respectively. The results showed that the expression level of wild-type human TXN was 100 mg / L, and that of the mutant was 120 mg / L. Regarding solubility, all samples were 100% soluble. The soluble component bands were clear in the electrophoresis images, and there was no significant protein accumulation in the inclusion body component, indicating that the protein was predominantly in soluble form and no inclusion bodies were formed, thus preserving its activity potential.

[0035] Table 2 Protein expression levels and soluble proportions

[0036] Example 2: Molecular-level antioxidant properties of thioredoxin mutants The ABTS radical scavenging ability of thioredoxin and its mutants was tested using the Total Antioxidant Capacity Assay Kit (ABTS Method) (S0119, Beyotime). ABTS radicals appear dark green. With the addition of substances with antioxidant properties, ABTS radicals can react with them, causing the reaction system to decolorize and turn pale blue, i.e., the absorbance decreases at 405 nm. Within a certain range, the change in absorbance is directly proportional to the degree of free radical scavenging.

[0037] Wild-type TXN and the mutant sr-(tripeptide-133 sh-peptide-2 hexapeptide-40) were prepared at the following concentration gradients (final concentrations): 2 μg / mL, 20 μg / mL, 200 μg / mL, 500 μg / mL, 1000 μg / mL, and 2000 μg / mL. After the addition of ABTS solution, the solution color faded to a pale blue to varying degrees. The data measured at 405 nm wavelength are shown in Table 3. The analysis shows that, at the molecular level, the mutant sr-(tripeptide-133 sh-peptide-2 hexapeptide-40) exhibited the highest antioxidant activity, especially in the concentration range of 200–2000 μg / mL. At a concentration of 200 μg / mL, the ABTS clearance rate of the mutant sr-(tripeptide-133 sh-peptide-2 hexapeptide-40) was 12%, which was 4.1% higher than that of the wild-type TXN; at a concentration of 2000 μg / mL, the ABTS clearance rate of the mutant sr-(tripeptide-133 sh-peptide-2 hexapeptide-40) reached 52.6%, which was 27.4% higher than that of the wild-type TXN.

[0038] Table 3. ABTS free radical scavenging rate (%) of each active ingredient

[0039] Example 3 An anti-aging composition, by weight percentage, comprises the following components: mutant sr-(tripeptide-133 sh-polypeptide-2 hexapeptide-40) 0.1%, glyceryl glucoside 0.5%, hydroxypinazone retinate 0.05%, ceramide NP 0.2%, ceramide AP 0.1%, resveratrol 0.3%, hydrogenated lecithin 0.1%, 2,3-butanediol 30%, laurocapram 0.1%, xanthan gum 0.1%, acrylamide dimethyl taurate ammonium / VP copolymer 0.5%, 1,2-hexanediol 2%, and water to make up the balance.

[0040] Example 4 An anti-aging composition, by weight percentage, comprises the following components: mutant sr-(tripeptide-133 sh-polypeptide-2 hexapeptide-40) 0.1%, glyceryl glucoside 0.5%, hydroxypinazone retinate 0.05%, ceramide NP 0.2%, ceramide AP 0.1%, resveratrol 0.3%, hydrogenated lecithin 0.5%; 2,3-butanediol 30%; laurocapram 0.1%, xanthan gum 0.1%, acrylamide dimethyl taurate ammonium / VP copolymer 0.5%, 1,2-hexanediol 2%, and water to make up the balance.

[0041] Example 5 An anti-aging composition, by weight percentage, comprises the following components: mutant sr-(tripeptide-133 sh-polypeptide-2 hexapeptide-40) 0.1%, glyceryl glucoside 0.5%, hydroxypinazone retinate 0.05%, ceramide NP 0.2%, ceramide AP 0.1%, resveratrol 0.3%, hydrogenated lecithin 1.0%; 2,3-butanediol 30%; laurocapram 0.1%, xanthan gum 0.1%, acrylamide dimethyl taurate ammonium / VP copolymer 0.5%, 1,2-hexanediol 2%, and water to make up the balance.

[0042] Example 6 An anti-aging composition, by weight percentage, comprises the following components: mutant sr-(tripeptide-133 sh-polypeptide-2 hexapeptide-40) 0.1%, glyceryl glucoside 0.5%, hydroxypinazone retinate 0.05%, ceramide NP 0.2%, ceramide AP 0.1%, resveratrol 0.3%, hydrogenated lecithin 1.5%; 2,3-butanediol 30%; laurocapram 0.1%, xanthan gum 0.1%, acrylamide dimethyl taurate ammonium / VP copolymer 0.5%, 1,2-hexanediol 2%, and water to make up the balance.

[0043] Comparative Example 1 An anti-aging composition, by weight percentage, comprises the following components: mutant sr-(tripeptide-133 sh-peptide-2 hexapeptide-40) 0.1%, glyceryl glucoside 0.5%, hydroxypinazone retinate 0.05%, ceramide NP 0.2%, ceramide AP 0.1%, resveratrol 0.3%, xanthan gum 0.1%, acrylamide dimethyl taurate ammonium / VP copolymer 0.5%, 1,2-hexanediol 2%, and water to make up the balance.

[0044] Preparation Example The composition and proportions are shown in Table 4. The preparation method includes the following steps: Step 1: Mix component B uniformly at 70℃ to obtain solution A; Step 2: Mix component C uniformly at 70℃ to obtain solution B; Step 3: Mix component C until homogeneous at room temperature to obtain solution C; Step 4: Mix solutions A and B at 70°C until homogeneous, homogenize at 6500 RPM for 3 minutes using a homogenizer, cool to 45°C, add solution C, mix until homogeneous, and cool to room temperature to obtain the anti-aging composition.

[0045] Table 4. Component Tables for Examples 3-6 and Comparative Example 1

[0046] To demonstrate the penetration-enhancing effect, transdermal absorption tests were conducted on Examples 3-6 and Comparative Example 1.

[0047] Using pig skin as a model, in vitro skin permeability tests were conducted in a Franz diffusion cell (reference standard: GB-T27818-20114 In vitro test method for skin absorption of chemicals). The cumulative transdermal rate of the mutant sr-(tripeptide-133 sh-polypeptide-2 hexapeptide-40) in the anti-aging composition at various time points was detected to evaluate the effect of the permeation-enhancing system on the transdermal performance of the anti-aging composition.

[0048] The test results of Examples 3-6 and Comparative Example 1 are as follows: Figure 3 ,Depend on Figure 3 It can be seen that the permeation-enhancing system improves the transdermal performance of the active ingredient, and Examples 5 and 6 have the highest permeation rates. Considering cost, the permeation-enhancing combination in Example 5 is the optimal result.

[0049] Example 7 An anti-aging composition, by weight percentage, comprises the following components: mutant sr-(tripeptide-133 sh-polypeptide-2 hexapeptide-40) 0.01%, glyceryl glucoside 0.5%, hydroxypinazone retinate 0.05%, ceramide NP 0.2%, ceramide AP 0.1%, resveratrol 0.3%, hydrogenated lecithin 1%; 2,3-butanediol 30%; laurocapram 0.1%, xanthan gum 0.1%, acrylamide dimethyl taurate ammonium / VP copolymer 0.5%, 1,2-hexanediol 2%, and water to make up the balance.

[0050] Example 8 An anti-aging composition, by weight percentage, comprises the following components: mutant sr-(tripeptide-133 sh-polypeptide-2 hexapeptide-40) 0.05%, glyceryl glucoside 0.5%, hydroxypinazone retinate 0.05%, ceramide NP 0.2%, ceramide AP 0.1%, resveratrol 0.3%, hydrogenated lecithin 1%; 2,3-butanediol 30%; laurocapram 0.1%, xanthan gum 0.1%, acrylamide dimethyl taurate ammonium / VP copolymer 0.5%, 1,2-hexanediol 2%, and water to make up the balance.

[0051] Example 9 An anti-aging composition, by weight percentage, comprises the following components: mutant sr-(tripeptide-133 sh-polypeptide-2 hexapeptide-40) 0.2%, glyceryl glucoside 0.5%, hydroxypinazone retinate 0.05%, ceramide NP 0.2%, ceramide AP 0.1%, resveratrol 0.3%, hydrogenated lecithin 1%; 2,3-butanediol 30%; laurocapram 0.1%, xanthan gum 0.1%, acrylamide dimethyl taurate ammonium / VP copolymer 0.5%, 1,2-hexanediol 2%, and water to make up the balance.

[0052] Comparative Example 2 An anti-aging composition, by weight percentage, comprises the following components: 0.2% thioredoxin, 0.5% glyceryl glucoside, 0.05% hydroxypinazone retinate, 0.2% ceramide NP, 0.1% ceramide AP, 0.3% resveratrol, 1% hydrogenated lecithin; 30% 2,3-butanediol; 0.1% laurocapram, 0.1% xanthan gum, 0.5% acrylamide dimethyl taurate ammonium / VP copolymer, 2% 1,2-hexanediol, and water to make up the balance.

[0053] Comparative Example 3 An anti-aging composition, by weight percentage, comprises the following components: 0.5% glyceryl glucoside, 0.05% hydroxypinazone retinate, 0.2% ceramide NP, 0.1% ceramide AP, 0.3% resveratrol, 1% hydrogenated lecithin; 30% 2,3-butanediol; 0.1% laurocapram, 0.1% xanthan gum, 0.5% acrylamide dimethyl taurate ammonium / VP copolymer, 2% 1,2-hexanediol, and water to make up the balance.

[0054] Comparative Example 4 An anti-aging composition, by weight percentage, comprises the following components: mutant sr-(tripeptide-133 sh-polypeptide-2 hexapeptide-40) 0.2%, hydrogenated lecithin 1%; 2,3-butanediol 30%; laurocapram 0.1%, xanthan gum 0.1%, acrylamide dimethyl taurate ammonium / VP copolymer 0.5%, 1,2-hexanediol 2%, and water to make up the balance.

[0055] Table 5. Component Tables for Examples 7-9 and Comparative Examples 2-4

[0056] The above-mentioned anti-aging composition can be used to prepare anti-aging cosmetics. The anti-aging cosmetic uses the above-mentioned anti-aging composition as an active ingredient, which is directly added to a cosmetic matrix; the amount added can be adjusted according to actual needs.

[0057] Example 10 The compositions (Examples 7-9, Comparative Examples 2-4) were added to the base serums, with the compositions comprising 2% of the total mass of the serums by weight. The serum formulations are shown in Table 6.

[0058] Table 6. Serum formulations containing 2% composition

[0059] Example 11 Skin irritation: Twenty volunteers aged 18-60 years were selected. Irritation tests were conducted using the human skin patch test according to the 2015 edition of the "Cosmetic Safety Technical Specifications". Samples 1-7 were added to the patch applicator, and then the applicator was applied to the inside of the subject's arm with non-irritating adhesive tape. The applicator was gently pressed with the palm to ensure even application to the skin. After 24 hours, the patch applicator was removed, and any residue was gently wiped away with a moistened cotton ball. Skin reaction was observed 0.5 hours later, after the indentation disappeared. If the result was negative, a second observation was conducted 24 hours after patch removal. The experimental results are shown in Table 7.

[0060] Table 7. Results of human irritation of serums containing 2% composition.

[0061] Example 12 To evaluate the anti-aging function of the composition on the skin, this application also evaluates the anti-aging efficacy of the composition by reducing the length of crow's feet.

[0062] Test Method: Twenty-one volunteers aged 20-50 were selected and tested under relatively stable environmental conditions with a temperature of 20℃-25℃ and humidity of 40%-60%. The 21 participants used samples 1-7 (see Example 10 for details), with three participants using one serum product each time. Before each use of the corresponding test sample, the participants washed their face with water, then applied the corresponding test sample twice daily (morning and evening) for two weeks.

[0063] Length of crow's feet wrinkles: Products or formulations with the same efficacy were prohibited from being used on the test area. The testing period was days 0, 7, and 14 after the tester used the sample. Before and after images were taken using VISIA7. Wrinkle parameters were analyzed by comparing the images to obtain the length of the crow's feet wrinkles, expressed in mm. A smaller value is better, indicating a lighter wrinkle, and is mainly used to determine the product's anti-wrinkle effect on the skin. The results are shown in Table 8 (average values ​​from the tests).

[0064] Table 8 Results of the eye wrinkle test after using the sample

[0065] The serums containing the compositions of Examples 7-9 and Comparative Examples 2-4 were tested for skin hydration and wrinkle length. Table 7 shows that the serum of Sample 3 (Example 9: a composition of mutant sr-(tripeptide-133 sh-polypeptide-2 hexapeptide-40)) exhibited superior wrinkle reduction compared to Sample 5 (Comparative Example 2: a composition of thioredoxin): a higher wrinkle reduction rate and noticeable effects within 7 days. The serums of Samples 1-3 (with added anti-aging compositions) showed progressively improved wrinkle reduction performance compared to Sample 7 (without added anti-aging compositions), with Samples 2-3 showing particularly significant improvement. The serum of Sample 3 was significantly more effective in reducing wrinkles than the serums of Samples 6-7.

[0066] The above results indicate that the composition of this application can improve skin wrinkles through a synergistic effect, when some When a component is missing, the efficacy of the composition is significantly reduced.

Claims

1. An anti-aging composition, characterized in that, By mass percentage, it consists of 0.01%-0.2% mutant sr-(tripeptide-133 sh-polypeptide-2 hexapeptide-40), 0.1%-1.0% glyceryl glucoside, 0.01%-0.15% hydroxypinazone retinate, 0.05%-0.5% ceramide NP, 0.05%-0.5% ceramide AP, 0.1%-0.5% resveratrol, and 97.15%-99.68% basic components; wherein, the basic components include water, thickener, preservative, and penetration enhancer.

2. The anti-aging composition according to claim 1, characterized in that, The basic components include 0.5-3% preservative, 0.1-1.5% thickener, 10-40% penetration enhancer, and water to make up the balance; the content of the basic components is 98.45%-99.84%, preferably 98.65%-98.84%, and most preferably 98.65%-98.8%.

3. The anti-aging composition according to claim 1, characterized in that, The anti-aging composition comprises 0.05%-0.2% of mutant sr-(tripeptide-133 sh-polypeptide-2 hexapeptide-40), 0.5% of glyceryl glucoside, 0.05% of hydroxypinazone retinate, 0.2% of ceramide NP, 0.1% of ceramide AP, 0.3% of resveratrol, and 98.65%-98.8% of basic components; wherein the basic components include 2% preservative, 0.6% thickener, 30.6%-31.6% penetration enhancer, and water to make up the balance.

4. The anti-aging composition according to claim 1, characterized in that, The preservative is selected from one of 1,2-hexanediol, pentanediol, p-hydroxyacetophenone, and phenoxyethanol; the thickener is selected from one or more of xanthan gum, acrylamide dimethyl taurate ammonium / VP copolymer, carbomer, and hydroxyethyl cellulose.

5. The anti-aging composition according to claim 1, characterized in that, The preservative is 1,2-hexanediol; the thickener is a combination of xanthan gum and acrylamide dimethyl taurate ammonium / VP copolymer, with the ratio of xanthan gum to acrylamide dimethyl taurate ammonium / VP copolymer being 1:1 to 1:10 by mass percentage.

6. The anti-aging composition according to claim 1, characterized in that, The penetration enhancer is one or more of hydrogenated lecithin, 2,3-butanediol, laurocapram, 1,3-butanediol, 1,3-propanediol, and polysorbate; preferably, it includes hydrogenated lecithin, 2,3-butanediol, and laurocapram, wherein, by mass percentage, hydrogenated lecithin accounts for 0.1-1.5% of the total mass of the composition; 2,3-butanediol accounts for 30% of the total mass of the composition; and laurocapram accounts for 0.1% of the total mass of the composition.

7. The anti-aging composition according to claim 1, characterized in that, The mutant is a thioredoxin mutant sr-(tripeptide-133 sh-polypeptide-2 hexapeptide-40), and its amino acid sequence is shown in SEQ ID NO:

4.

8. A method for preparing the anti-aging composition according to any one of claims 1-7, characterized in that, Includes the following steps: Step 1: Mix the prescribed amounts of hydroxypinazone retinate, ceramide NP, ceramide AP, resveratrol, 2,3-butanediol, and laurocapram at 70-80℃ to obtain solution A; Step 2: Mix the prescribed amounts of hydrogenated lecithin, xanthan gum, acrylamide dimethyl taurate ammonium / VP copolymer, 1,2-hexanediol and water at 70-80℃ until homogeneous to obtain solution B; Step 3: Mix the prescribed amounts of mutant sr-(tripeptide-133 sh-polypeptide-2 hexapeptide-40) and glycerol glucoside at room temperature to obtain solution C; Step 4: Mix solutions A and B at 70-80℃ until homogeneous, homogenize with a homogenizer, cool to 40-45℃, add solution C, mix until homogeneous, and obtain the anti-aging composition.

9. The use of any of the anti-aging compositions according to claims 1-7 in the preparation of skin care products or personal care products, wherein the skin care product is a serum, cream, lotion or mask; and the personal care product is a facial cleanser, shower gel or body lotion.

10. An essence, characterized in that, Includes the anti-aging composition according to any one of claims 1-7.

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