An aspartate peptide mixture, its preparation method and use in the preparation of skin aging delaying products

Abstract

This invention relates to an asparagine peptide mixture, its preparation method, and its application in the preparation of products that delay skin aging, belonging to the field of cosmetic technology. The asparagine peptide mixture provided by this invention is an active peptide mixture with a molecular weight <5 kDa, prepared from asparagus root tubers via enzymatic hydrolysis. This asparagine peptide mixture can significantly enhance the vitality of human skin fibroblasts (HSF), promote cell proliferation, and enhance the tissue repair capacity of HSF cells; it can effectively inhibit β-galactosidase activity while increasing the expression level of pre-type I collagen. Furthermore, this invention identifies the top ten most active short peptides in the asparagine peptide mixture through structural identification and activity screening, and demonstrates through molecular docking that the asparagine peptide mixture intervenes in the skin aging process through multiple pathways, providing solid in vitro scientific evidence for the development of natural peptide cosmetics with multiple anti-aging effects from the traditional medicinal plant asparagus.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, and in particular to an aspartic peptide mixture, its preparation method, and its application in the preparation of products that delay skin aging. Background Technology

[0002] Aging is an inevitable process of life, influenced by multiple factors including the external environment and the internal gene regulatory network. It ultimately manifests as wrinkle formation, decreased elasticity, weakened barrier function, and a decline in self-repair capabilities. Human skin fibroblasts are the main cells synthesizing collagen, elastin, and other extracellular matrix components. With age or a surge in oxidative stress, their proliferative capacity declines, and their function is impaired, leading to age-related secretory phenotypes and resulting in skin laxity. In recent years, bioactive peptides, due to their well-defined cell signaling regulatory functions, have become an important direction in anti-aging cosmetic research. Therefore, discovering peptide complexes with multi-target activities from natural plants is gradually becoming a new trend in green and sustainable skincare research and development. Asparagus (Asparagus officinalis) is a plant belonging to the genus Asparagus in the family Liliaceae. Asparagus cochinchinensis Asparagus root (Lour.) Merr.) has the effects of nourishing yin and moisturizing dryness, clearing the lungs and reducing fire. At present, the research on the activity of asparagus is mostly focused on polysaccharides and saponins. There are currently no reports on the anti-aging effects of peptides derived from asparagus. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide an asparagine mixture, its preparation method, and its application in the preparation of products that delay skin aging.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides an asparagine mixture, wherein the asparagine mixture is an active peptide mixture with a molecular weight of <5 kDa obtained by enzymatic hydrolysis of asparagus root as raw material.

[0005] This invention utilizes an optimized enzymatic hydrolysis process to extract a mixture of natural active peptides—aspartic peptides—from asparagus. A series of in vitro cell experiments were conducted to systematically evaluate the anti-aging bioactivity of this asparagus-derived peptide mixture. Results showed that the aspartic peptide mixture significantly enhanced the viability of human skin fibroblasts (HSF) (p < 0.05), promoted cell proliferation, and strengthened the tissue repair capacity of HSF. In an H2O2-induced cell senescence model, the aspartic peptide mixture exhibited a significant anti-damage effect, effectively inhibiting β-galactosidase activity while simultaneously increasing the expression level of pre-type I collagen.

[0006] Furthermore, the molecular weight of the aspartic peptide mixture is <1 kDa.

[0007] Furthermore, the asparagine mixture may include, but is not limited to, short peptides with the following amino acid sequences: FF, FW, WFW, MPF, FFPF, FPF, WPF, WFL, LPF, FPL, LFP, FP, GFL, FLG, LGF, APLF; The amino acid sequence is represented by a single-letter amino acid code, where F represents phenylalanine (Phe), W represents tryptophan (Trp), P represents proline (Pro), L represents leucine (Leu), M represents methionine (Met), G represents glycine (Gly), and A represents alanine (Ala).

[0008] This invention further screens for a mixture of asparagine peptides with smaller molecular weights (<1 kDa), which exhibit stronger anti-aging activity. Furthermore, the smaller molecular weight also enhances skin affinity. The composition of the asparagine peptide mixture is analyzed using LC-MS. Subsequently, PeptideRanker is used to predict peptide activity, screening for peptides with an activity score greater than 0.5. Short peptides with high activity and high content (FF, FW, WFW, MPF, FFPF, FPF, WPF, WFL, LPF, FPL, LFP, FP, GFL, FLG, LGF, APLF) are further screened and molecular docking simulations are performed. The results show that these short peptides can bind to Sirt2, ERK1, and mTOR targets, indicating that the asparagine peptide mixture intervenes in the skin aging process through multiple pathways. This provides a solid in vitro scientific basis for developing cosmetics containing natural peptides with multiple anti-aging effects from the traditional medicinal plant asparagus.

[0009] Furthermore, the preparation method of the asparagine mixture includes the following steps: S1. Crush the dried asparagus tubers to obtain asparagus powder, add pure water, stir and soak at 4-10℃ for 20-50 minutes, and treat with pulse ultrasound to obtain pretreated material; S2. Adjust the pH of the pretreated material to 7.2-8.5, add trypsin, carry out the first stirring reaction, filter with a filter cloth to obtain filter residue and the first peptide filtrate; take the filter residue, add pure water, adjust the pH to 6.2-7.5, then add papain and neutral protease, carry out the second stirring reaction, filter with a filter cloth to obtain the second peptide filtrate; combine the two peptide filtrates, inactivate the enzymes, filter by suction to obtain a clear filtrate; S3. Concentrate the clarified filtrate, precipitate it with ethanol, and centrifuge to obtain the supernatant. After removing the ethanol from the supernatant, use an ultrafiltration membrane system to retain the permeate with a molecular weight <5KDa and collect the permeate. S4. The permeate is freeze-dried under vacuum to obtain a mixture of aspartic peptides.

[0010] Preferably, the amount of pure water added in step S1 is 5-10 times the mass of the asparagus powder.

[0011] Preferably, the stirring and soaking speed in step S1 is 300-500 r / min.

[0012] Preferably, the conditions for pulsed ultrasound treatment in step S1 are: power 300W, frequency 20~40kHz, pulse mode of 3s working and 3s intermittent, and ultrasound treatment time of 10-30min.

[0013] Preferably, the amount of trypsin added in step S2 is 1%-4% of the mass of asparagus powder, and the temperature of the first stirring reaction is 45-55℃, the rotation speed is 300-500 r / min, and the time is 60 min.

[0014] Preferably, the amount of pure water added in step S2 is 5-10 times the mass of the asparagus powder.

[0015] Preferably, in step S2, the amount of papain added is 1%-2% of the mass of asparagus powder, the amount of neutral protease added is 0.5%-1.5% of the mass of asparagus powder, and the temperature of the second stirring reaction is 45-55℃, the rotation speed is 300-500 r / min, and the time is 60 min.

[0016] Preferably, the filter cloth filtration in step S2 is filtration with a 100-mesh filter cloth, the enzyme inactivation is carried out by maintaining the filter cloth in a water bath at 80-90℃ for 8-10 minutes, and the vacuum filtration is carried out using a Buchner funnel with a 0.45μm microporous filter membrane.

[0017] Preferably, the concentration in step S3 is vacuum concentration to 1 / 5 to 1 / 10 of the original volume.

[0018] Preferably, the ethanol precipitation in step S3 is carried out by slowly adding 95% v / v ethanol to the concentrate while stirring at a speed of 120-150 r / min until the final ethanol concentration in the system reaches 85% v / v, and then letting it stand at 4°C for 10-16 h.

[0019] Preferably, the centrifugation in step S3 is performed at a speed of 8000-9000 r / min for 20-30 min, and the removal of ethanol from the supernatant is performed by vacuum concentration at room temperature.

[0020] Preferably, in step S3, an ultrafiltration membrane system is used to retain permeate with a molecular weight <1 kDa.

[0021] Secondly, the present invention provides the use of the asparagine mixture described in the first aspect in the preparation of products for delaying skin aging.

[0022] Furthermore, the effective concentration of the asparagine mixture in the product is not less than 1.0 mg / mL.

[0023] Preferably, the effective concentration of the asparagine mixture in the product is 1.0-12.5 mg / mL, more preferably 3-12.5 mg / mL.

[0024] Furthermore, the products include cosmetics.

[0025] Thirdly, the present invention provides a cosmetic product for delaying skin aging, comprising the asparagine mixture described in the first aspect.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention extracts a mixture of natural active peptides—asparagine peptides—from asparagus through an optimized enzymatic hydrolysis process. The asparagine peptide mixture can significantly enhance the vitality of human skin fibroblasts (HSF), promote cell proliferation, and enhance the ability of HSF cells to repair tissues; it can effectively inhibit β-galactosidase activity while increasing the expression level of pre-type I collagen.

[0027] This invention screened out the most active short peptides in the asparagine peptide mixture: FF, FW, WFW, MPF, FFPF, FPF, WPF, WFL, LPF, FPL, LFP, FP, GFL, FLG, LGF, and APLF. Molecular docking showed that these short peptides can bind to the targets of Sirt2, ERK1, and mTOR, indicating that the asparagine peptide mixture intervenes in the skin aging process through multiple pathways. This provides a solid in vitro scientific basis for the development of natural peptide cosmetics with multiple anti-aging effects from the traditional medicinal plant asparagus. Attached Figure Description

[0028] Figure 1 The results show the molecular docking of the aspartic peptide mixture with the targets Sirt2, ERK1, and mTOR. Detailed Implementation

[0029] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0030] The materials used in the following embodiments are from the following sources: Trypsin: Purchased from Fuzhou Feijing Biotechnology Co., Ltd., product number: PH9338, enzyme activity: ≥200000U / mg; Papain: Purchased from Beijing Innocare Technology Co., Ltd., product number: B99890, enzyme activity: ≥6000U / mg; Neutral protease: purchased from Fuzhou Feijing Biotechnology Co., Ltd., product number: PH9339, enzyme activity: ≥100000U / mg.

[0031] Unless otherwise specified, all other materials and reagents used in the examples are commercially available.

[0032] Example 1 This embodiment provides a method for preparing an aspartic peptide mixture, the steps of which are as follows: S1. Crush the dried asparagus tubers to obtain asparagus powder, add pure water, stir and soak at 4℃ for 35 minutes, and treat with pulsed ultrasound to obtain pretreated material; S2. Adjust the pH of the pretreated material to 8.0, add trypsin, carry out the first stirring reaction, filter with a filter cloth to obtain filter residue and the first peptide filtrate; take the filter residue, add pure water, adjust the pH to 6.5, then add papain and neutral protease, carry out the second stirring reaction, filter with a filter cloth to obtain the second peptide filtrate; combine the two peptide filtrates, inactivate the enzymes, filter by suction to obtain a clear filtrate; S3. Concentrate the clarified filtrate, precipitate it with ethanol, and centrifuge to obtain the supernatant. After removing the ethanol from the supernatant, use an ultrafiltration membrane system to retain the permeate with a molecular weight <1 kDa and collect the permeate. S4. The permeate is freeze-dried under vacuum to constant weight to obtain aspartic peptide mixture 1; In step S1, the amount of pure water added is 8 times the mass of the asparagus powder; the stirring and soaking speed is 400 r / min; and the pulse ultrasonic treatment conditions are: power 300W, frequency 30kHz, pulse mode of 3s working, 3s intermittent, and total ultrasonic treatment time of 20min. In step S2, the amount of trypsin added is 2.5% of the mass of the asparagus powder; the temperature of the first stirring reaction is 50℃, the speed is 400 r / min, and the time is 60min; the amount of pure water added is 8 times the mass of the asparagus powder; the amount of papain added is 1.5% of the mass of the asparagus powder; the amount of neutral protease added is 1% of the mass of the asparagus powder; and the temperature of the second stirring reaction is... The temperature is 50℃, the rotation speed is 400r / min, and the time is 60min. The filter cloth filtration is performed using a 100-mesh filter cloth. The enzyme inactivation is performed by maintaining the solution in an 85℃ water bath for 9min. The vacuum filtration is performed using a Buchner funnel with a 0.45μm microporous membrane. In step S3, the concentration is performed by vacuum concentration at room temperature to 1 / 8 of the original volume. The ethanol precipitation is performed by slowly adding 95% v / v ethanol to the concentrate while stirring at 130r / min until the final ethanol concentration in the system reaches 85% v / v, and then letting it stand at 4℃ for 12h. The centrifugation is performed by centrifugation at 8500r / min for 25min. The removal of ethanol from the supernatant is performed by vacuum concentration at room temperature.

[0033] Example 2 This embodiment provides a method for preparing an aspartic peptide mixture, the steps of which are as follows: S1. Crush the dried asparagus tubers to obtain asparagus powder, add pure water, stir and soak at 4℃ for 20 minutes, and treat with pulsed ultrasound to obtain pretreated material; S2. Adjust the pH of the pretreated material to 7.2, add trypsin, carry out the first stirring reaction, filter with a filter cloth to obtain filter residue and the first peptide filtrate; take the filter residue, add pure water, adjust the pH to 6.2, then add papain and neutral protease, carry out the second stirring reaction, filter with a filter cloth to obtain the second peptide filtrate; combine the two peptide filtrates, inactivate the enzymes, filter by suction to obtain a clear filtrate; S3. Concentrate the clarified filtrate, precipitate it with ethanol, and centrifuge to obtain the supernatant. After removing the ethanol from the supernatant, use an ultrafiltration membrane system to retain the permeate with a molecular weight <5KDa and collect the permeate. S4. The permeate is freeze-dried under vacuum to constant weight to obtain aspartic peptide mixture 2; In step S1, the amount of pure water added is 5 times the mass of the asparagus powder; the stirring and soaking speed is 500 r / min; and the pulsed ultrasonic treatment conditions are: power 300W, frequency 20kHz, pulse mode of 3s working, 3s intermittent, and total ultrasonic treatment time of 30min. In step S2, the amount of trypsin added is 1% of the mass of the asparagus powder; the temperature of the first stirring reaction is 45℃, the speed is 300 r / min, and the time is 60min; the amount of pure water added is 5 times the mass of the asparagus powder; the amount of papain added is 1% of the mass of the asparagus powder; the amount of neutral protease added is 0.5% of the mass of the asparagus powder; and the temperature of the second stirring reaction is... The process involves: filtration at 45°C, rotation speed of 300 r / min, and time of 60 min; filtration using a 100-mesh filter cloth; enzyme inactivation by maintaining the solution in an 80°C water bath for 10 min; and vacuum filtration using a Buchner funnel fitted with a 0.45 μm microporous membrane. In step S3, concentration is achieved by vacuum concentration at room temperature to 1 / 5 of the original volume. Ethanol precipitation involves slowly adding 95% v / v ethanol to the concentrate while stirring at 120 r / min until the final ethanol concentration reaches 85% v / v, followed by standing at 4°C for 12 h. Centrifugation is performed at 9000 r / min for 20 min. Ethanol removal from the supernatant is achieved by vacuum concentration at room temperature.

[0034] Example 3 This embodiment provides a method for preparing an aspartic peptide mixture, the steps of which are as follows: S1. Crush the dried asparagus tubers to obtain asparagus powder, add pure water, stir and soak at 4℃ for 50 minutes, and treat with pulsed ultrasound to obtain pretreated material; S2. Adjust the pH of the pretreated material to 8.5, add trypsin, carry out the first stirring reaction, filter with a filter cloth to obtain filter residue and the first peptide filtrate; take the filter residue, add pure water, adjust the pH to 7.5, then add papain and neutral protease, carry out the second stirring reaction, filter with a filter cloth to obtain the second peptide filtrate; combine the two peptide filtrates, inactivate the enzymes, filter by suction to obtain a clear filtrate; S3. Concentrate the clarified filtrate, precipitate it with ethanol, and centrifuge to obtain the supernatant. After removing the ethanol from the supernatant, use an ultrafiltration membrane system to retain the permeate with a molecular weight <1 kDa and collect the permeate. S4. The permeate is freeze-dried under vacuum to constant weight to obtain aspartic peptide mixture 1; In step S1, the amount of pure water added is 10 times the mass of the asparagus powder; the stirring and soaking speed is 300 r / min; and the pulse ultrasonic treatment conditions are: power 300W, frequency 40kHz, pulse mode of 3s working, 3s intermittent, and total ultrasonic treatment time of 10min. In step S2, the amount of trypsin added is 4% of the mass of the asparagus powder; the temperature of the first stirring reaction is 55℃, the speed is 500 r / min, and the time is 60min; the amount of pure water added is 10 times the mass of the asparagus powder; the amount of papain added is 2% of the mass of the asparagus powder; the amount of neutral protease added is 1.5% of the mass of the asparagus powder; and the temperature of the second stirring reaction is... The process involves the following steps: 55°C, 500 rpm, and 60 min; filtration using a 100-mesh filter cloth; enzyme inactivation in a 90°C water bath for 8 min; vacuum filtration using a Buchner funnel with a 0.45 μm microporous membrane; concentration in step S3 by vacuum concentration at room temperature to 1 / 10 of the original volume; ethanol precipitation by slowly adding 95% v / v ethanol to the concentrate while stirring at 150 rpm until the final ethanol concentration reaches 85% v / v, followed by standing at 4°C for 12 h; centrifugation at 8000 rpm for 30 min; and removal of ethanol from the supernatant by vacuum concentration at room temperature.

[0035] Comparative Example 1 This comparative example provides a method for preparing an asparagine mixture, which differs from Example 1 in step S1. Specifically, the dried asparagus root is crushed to obtain asparagus powder, and pure water with a mass of 10 times that of the asparagus powder is added. The mixture is stirred and soaked at room temperature for 55 minutes to obtain a pretreated material. The remaining steps and parameters are the same as in Example 1, and asparagine mixture 1' is prepared.

[0036] Comparative Example 2 This comparative example provides a method for preparing an asparagine mixture, which differs from Example 1 in step S2, specifically as follows: The pH of the pretreated material was adjusted to 6.5, papain and neutral protease were added, and the first stirring reaction was carried out. The mixture was then filtered through a filter cloth to obtain a filter residue and a first peptide filtrate. The filter residue was added to pure water, the pH was adjusted to 8.0, and trypsin was added. The mixture was then stirred and filtered through a filter cloth to obtain a second peptide filtrate. The two peptide filtrates were combined, the enzymes were inactivated, and the mixture was filtered to obtain a clear filtrate. The papain was added at 1.5% of the mass of asparagus powder, the neutral protease at 1% of the mass of asparagus powder, and the trypsin at 2.5% of the mass of asparagus powder. The first stirring reaction was carried out at 50°C, 400 rpm, and 60 min. The amount of pure water added was 8 times the mass of asparagus powder. The second stirring reaction was carried out at 50°C, 400 rpm, and 60 min. The enzyme inactivation was performed by maintaining the solution in an 85°C water bath for 9 min. The filtration was performed using a Buchner funnel fitted with a 0.45 μm microporous membrane. The remaining steps and parameters are the same as in Example 1, and asparagine mixture 2' is prepared.

[0037] Comparative Example 3 This comparative example provides a method for preparing an asparagine mixture, which differs from Example 1 in step S2, specifically: The pH of the pretreated material was adjusted to 6.5, papain and neutral protease were added, the mixture was stirred and reacted, and then filtered through a filter cloth to obtain a peptide filtrate. The peptide filtrate was then deactivated by enzyme inactivation and filtered to obtain a clear filtrate. The amount of papain added is 3% of the mass of asparagus powder, the amount of neutral protease added is 2% of the mass of asparagus powder, the stirring reaction temperature is 50℃, the stirring speed is 400r / min, and the time is 120min, the filter cloth filtration is performed using a 100-mesh filter cloth, the enzyme inactivation is performed by maintaining the solution in an 85℃ water bath for 9min, and the vacuum filtration is performed using a Buchner funnel fitted with a 0.45μm microporous membrane. The remaining steps and parameters are the same as in Example 1, and asparagine mixture 3' is prepared.

[0038] Comparative Example 4 This comparative example provides a method for preparing an asparagine mixture, which differs from Example 1 in step S2, specifically as follows: The pH of the pretreated material was adjusted to 8.0, trypsin was added, the mixture was stirred and reacted, and then filtered through a filter cloth to obtain a peptide filtrate. The peptide filtrate was then deactivated by enzyme inactivation and filtered to obtain a clear filtrate. The amount of trypsin added is 5% of the mass of asparagus powder; the stirring reaction temperature is 50℃, the stirring speed is 400r / min, and the time is 120min; the filter cloth filtration is performed using a 100-mesh filter cloth; the enzyme inactivation is performed by maintaining the solution in an 85℃ water bath for 9min; and the vacuum filtration is performed using a Buchner funnel fitted with a 0.45μm microporous membrane. The remaining steps and parameters are the same as in Example 1, and the aspartic peptide mixture 4' is prepared.

[0039] Comparative Example 5 This comparative example provides a method for preparing an asparagine mixture, which differs from Example 1 in step S2, specifically as follows: The pH of the pretreated material was adjusted to 8.0, trypsin was added, and the first stirring reaction was carried out. The mixture was then filtered through a filter cloth to obtain a filter residue and a first peptide filtrate. The filter residue was added to pure water, the pH was adjusted to 6.5, papain was added, and the second stirring reaction was carried out. The mixture was then filtered through a filter cloth to obtain a second peptide filtrate. The two peptide filtrates were combined, the enzyme was inactivated, and the mixture was filtered to obtain a clear filtrate. The following steps are described: the amount of trypsin added is 2.5% of the mass of asparagus powder; the temperature of the first stirring reaction is 50℃, the rotation speed is 400 r / min, and the time is 60 min; the amount of pure water added is 8 times the mass of asparagus powder; the amount of papain added is 2.5% of the mass of asparagus powder; the temperature of the second stirring reaction is 50℃, the rotation speed is 400 r / min, and the time is 60 min; the filter cloth filtration is performed using a 100-mesh filter cloth; the enzyme inactivation is performed by maintaining the solution in an 85℃ water bath for 9 min; and the vacuum filtration is performed using a Buchner funnel fitted with a 0.45 μm microporous membrane. The remaining steps and parameters are the same as in Example 1, and an aspartic peptide mixture 5' is prepared.

[0040] Comparative Example 6 This comparative example provides a method for preparing an asparagine mixture, which differs from Example 1 in that the final ethanol concentration of the ethanol precipitation in step S3 is 50% v / v. The remaining steps and parameters are the same as in Example 1, and an aspartic peptide mixture 6' is prepared.

[0041] Comparative Example 7 This comparative example provides a method for preparing an asparagine mixture, which differs from Example 1 in that: in step S3, an ultrafiltration membrane system is used to retain the permeate with a molecular weight <0.5 kDa; The remaining steps and parameters are the same as in Example 1, and an aspartic peptide mixture 7' is prepared.

[0042] Comparative Example 8 This comparative example provides a method for preparing an asparagine mixture, which differs from Example 1 in that: in step S3, an ultrafiltration membrane system is used to retain the permeate with a molecular weight <8 kDa; The remaining steps and parameters are the same as in Example 1, and an aspartic peptide mixture 8' is prepared.

[0043] The yield of the asparagine mixtures prepared in Examples 1-3 and Comparative Examples 1-8 was determined: the mass of the lyophilized asparagine mixture obtained in each example and comparative example was accurately weighed, and the yield (%) of the asparagine mixture was calculated as the percentage of the mass of the lyophilized powder to the initial mass of the corresponding batch of dried asparagus root. The yield calculation formula is as follows: Yield (%) = final mass of asparagine mixture (g) / initial mass of dried asparagus root (g) × 100%.

[0044] The results are shown in Table 1. The yields of the asparagine mixtures prepared in Examples 1-3 reached 8.30%-8.80%, which were higher than those in Comparative Examples 1-8. In Comparative Examples 1-8, the raw material pretreatment method (Comparative Example 1), enzymatic hydrolysis method (Comparative Examples 2-5), final ethanol concentration during alcohol precipitation (Comparative Example 6), and molecular weight cutoff of ultrafiltration membrane (Comparative Examples 7-8) were adjusted. This shows that the preparation steps of the present invention can extract peptides from asparagus tubers more efficiently, and have better application value and economic benefits.

[0045] Table 1. Yield and property evaluation results of the asparagine mixture Group Yield Product Color Example 1 8.80% White to pale yellow Example 2 8.46% White to pale yellow Example 3 8.37% White to pale yellow Comparative Example 1 5.44% White to pale yellow Comparative Example 2 7.93% White to pale yellow Comparative Example 3 7.05% White to pale yellow Comparative Example 4 6.38% White to pale yellow Comparative Example 5 6.05% White to pale yellow Comparative Example 6 8.04% Dark yellow Comparative Example 7 6.32% White to pale yellow Comparative Example 8 8.67% White to pale yellow Test Example 1: Effect of a mixture of aspartic peptide molecules on the viability of human skin fibroblasts (HSF) This test case investigated the effect of asparagine mixture on HSF cell proliferation. First, the effect of the asparagine mixture prepared in Example 1 at different concentrations on HSF cell proliferation was measured. Then, the effects of the asparagine mixtures prepared in Examples 1-3 and Comparative Examples 1-8 on HSF cell proliferation were compared.

[0046] Test Principle: The CCK-8 reagent contains WST-8 [chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt], which is reduced by dehydrogenases in the mitochondria of cells to a highly water-soluble yellow formazan product under the action of the electron carrier 1-methoxy-5-methylphenazine sulfate dimethyl (1-Methoxy PMS). The amount of formazan generated is directly proportional to the number of viable cells. The absorbance value is measured at 450 nm using a microplate reader, which indirectly reflects the number of viable cells and is used in cell proliferation assays. By measuring the absorbance values ​​after administration of the test substance and the blank control, the efficacy of the test substance in promoting cell proliferation is evaluated.

[0047] The cell line used was human skin fibroblasts (HSF) (Shanghai Cell Bank, Chinese Academy of Sciences). The testing conditions were: incubator temperature 37±1℃, humidity 90±5%, carbon dioxide 5±1%. Cells were cultured and treated according to groups, followed by testing. The testing methods are as follows: Sample preparation: (a) The asparagine mixture prepared in Example 1 was prepared into a stock solution of 50 mg / mL with PBS, and then diluted with serum-free DMEM medium (DME101500, Jetech) to prepare sample solutions with concentrations of 1.5625, 3.125, 6.25 and 12.5 mg / mL, respectively. Three parallel tubes were set up for each sample. (b) The asparagine mixtures prepared in Examples 1-3 and Comparative Examples 1-8 were prepared into a stock solution of 50 mg / mL with PBS, and then diluted with serum-free DMEM medium (DME101500, Jetech) to a sample solution with a concentration of 6.25 mg / mL. Three parallel tubes were set up for each sample. (2) The cell suspension was seeded into a 96-well cell culture plate at a density of 6000 cells / well. 100 μL of DMEM culture medium containing 10% v / v FBS was added to each well and cultured for 24 h. (3) Sample feeding: Discard the original culture medium of each group. Add 100 μL of serum-free DMEM medium (DME101500, Jete) to the control group and add 100 μL of sample solution to each sample group. Incubate for 24 h. (4) CCK-8 test: After 24 hours of culture, the original culture medium was discarded, and 100 μL of DMEM medium containing CCK-8 (B34304, Selleckchem) was added to each well (CCK-8 = 10:1). The culture was continued at 37±1℃ for 1 hour. The absorbance was measured at 450 nm, and the proliferation rate was calculated. Proliferation rate = (Sample group A - Control group A) / (Control group A - Blank group A) × 100%; Note: Blank A refers to the absorbance value of cell-free medium containing 100 μL of CCK-8, used to subtract the background effect of CCK-8; The higher the proliferation rate, the stronger the promoting effect of the aspartic polypeptide mixture on HSF cell proliferation.

[0048] The effects of the asparagine mixture prepared in Example 1 on the proliferation of HSF cells at different concentrations were averaged, as shown in Table 1. The proliferation rate of HSF cells at different concentrations of asparagine mixture showed a normal distribution, with the asparagine mixture at a concentration of 6.25 mg / mL showing the best proliferation-promoting effect on HSF cells.

[0049] Table 2. Results of the proliferation-promoting effect of different concentrations of asparagine mixtures on HSF cells. Concentration (mg / mL) Proliferation rate (%) 1.5625 6.18 3.125 32.06 6.25 73.49 12.5 50.03 The proliferation-promoting results of the asparagine peptide mixtures in Examples 1-3 and Comparative Examples 1-8 are averaged, as shown in Table 3. The asparagine peptide mixtures prepared in Examples 1-3 achieved a proliferation-promoting rate of 70.56%-73.49% on HSF cells, indicating that the asparagine peptide mixtures can effectively promote HSF cell proliferation. Comparing the data of Example 1 and Comparative Examples 1-8, it can be seen that the proliferation-promoting rate of HSF cells in Comparative Examples 1-8 is relatively low. Among them, the molecular weight cutoff of the ultrafiltration membrane (Comparative Examples 7-8) has the greatest impact on the HSF cell proliferation-promoting effect of the prepared asparagine peptide mixtures, indicating that the active substances in the asparagine peptide mixtures with a molecular weight <5 kDa work together to exert the best proliferation-promoting effect. Secondly, the raw material pretreatment step (Comparative Example 1), the enzymatic hydrolysis step (Comparative Examples 2-5), and the alcohol precipitation step (Comparative Example 6) also have a certain impact on the HSF cell proliferation-promoting effect of the asparagine peptide mixtures, indicating that the asparagine peptide mixtures prepared by selecting the steps specified in this invention have a better proliferation-promoting effect on HSF cells.

[0050] Table 3. Results of the proliferation-promoting effect of aspartic peptide mixture on HSF cells. Group Proliferation rate (%) Example 1 73.49 Example 2 70.56 Example 3 72.38 Comparative Example 1 62.60 Comparative Example 2 63.35 Comparative Example 3 52.09 Comparative Example 4 55.17 Comparative Example 5 57.21 Comparative Example 6 51.95 Comparative Example 7 40.48 Comparative Example 8 45.62 Test Example 2: Inhibitory effect of aspartic peptide mixture on oxidative damage in human skin fibroblasts (HSF) This test case investigates the inhibitory effect of a mixture of aspartic peptides on cellular oxidative damage. Hydrogen peroxide (H2O2), as a common exogenous oxidant, can induce human skin fibroblasts to produce reactive oxygen species, further damaging cells and affecting the number of viable cells and the activity of mitochondrial dehydrogenases. By adding exogenous antioxidants, oxidative damage to cells can be prevented and cell viability restored. The CCK-8 assay indirectly reflects the inhibitory effect of the test substance on cellular oxidative damage.

[0051] The cell line used was human skin fibroblasts (HSF) (Shanghai Cell Bank, Chinese Academy of Sciences). The testing conditions were: incubator temperature 37±1℃, humidity 90±5%, carbon dioxide 5±1%. Cells were cultured and treated according to groups, followed by testing. The testing methods are as follows: (1) Sample preparation: The samples (the mixture of asparagine prepared in Examples 1-3 and Comparative Examples 1-8) were prepared into a stock solution of 50 mg / mL with PBS, and then diluted with DMEM medium (DME101500, Jetech) containing 10% v / v FBS to a sample solution with a concentration of 6.25 mg / mL. (2) The cell suspension was seeded into a 96-well cell culture plate at a density of 10,000 cells / well. 100 μL of DMEM culture medium containing 10% v / v FBS was added to each well and cultured for 24 h. (3) Sample feeding: Discard the original culture medium in each group. Add 100 μL of DMEM medium (DME101500, Jete) containing 10% v / v FBS to the blank group and the control group respectively; add 100 μL of sample solution to each sample group. Each group treatment is set up in 3 parallel experiments. (4) Damage: After culturing for 0.5 h, except for the blank group, each group was given 50 μL of H2O2 (88597-100mL-F, Sigma) solution with a concentration of 3000 μmol / L. The blank group was given 50 μL of DMEM medium containing 10% v / v FBS. Each group was cultured for another 24 h. (4) CCK-8 test: After culturing for 24 hours, the original culture medium was discarded, and 100 μL of culture medium containing CCK-8 (B34304, Selleckchem) (DMEM medium: CCK-8 = 10:1) was added to each well. The culture was continued at 37±1℃ for 1 hour. The absorbance value was measured at a wavelength of 450 nm. Compared with the blank group, the absorbance value of the control group cells decreased significantly, indicating that the H2O2 oxidative damage model was successfully modeled.

[0052] Calculate the damage inhibition rate for each sample group: Damage inhibition rate = (OD value sample group - OD value control group) / OD value control group × 100%; The higher the damage inhibition rate, the stronger the ability of the polypeptide mixture to inhibit oxidative damage.

[0053] The results were averaged, as shown in Table 4. The asparagine mixtures prepared in Examples 1-3 achieved a proliferation rate of 52.41%-58.69% on HSF cells, indicating that the asparagine mixtures have a strong inhibitory ability against oxidative damage. Comparing the data of Example 1 with Comparative Examples 1-8, the damage inhibition rate of Comparative Examples 1-8 on HSF cells was much lower than that of Example 1, with Comparative Examples 7-8 showing the lowest damage inhibition rate. This indicates that during the preparation of the asparagine mixture, the molecular weight cutoff of the ultrafiltration membrane has the greatest impact on its ability to inhibit oxidative damage, and the active substances in the asparagine mixture with a molecular weight <5 kDa have the strongest ability to inhibit oxidative damage. Meanwhile, the raw material pretreatment step (Comparative Example 1), the enzymatic hydrolysis step (Comparative Examples 2-5), and the alcohol precipitation step (Comparative Example 6) all affect the ability of the asparagine mixture to inhibit oxidative damage.

[0054] Table 4 Results of the inhibitory effect of aspartic peptide mixture on HSF cells. Group Damage inhibition rate (%) Example 1 58.69 Example 2 52.41 Example 3 54.15 Comparative Example 1 40.07 Comparative Example 2 44.32 Comparative Example 3 33.48 Comparative Example 4 36.69 Comparative Example 5 37.25 Comparative Example 6 30.78 Comparative Example 7 24.62 Comparative Example 8 28.81 Test Example 3: Inhibitory effect of aspartic peptide mixture on aging of human skin fibroblasts (HSF) Hydrogen peroxide (H2O2), a common exogenous oxidant, can induce human skin fibroblasts to produce reactive oxygen species, further damaging cells and leading to cellular senescence. Adding substances that inhibit senescence can prevent cell senescence and restore cell vitality. β-galactosidase is a commonly used marker enzyme for detecting cellular senescence, especially senescence-associated β-galactosidase (SA-β-gal). A β-galactosidase staining kit is a kit that stains senescent cells or tissues based on the upregulation of senescence-associated β-galactosidase (SA-β-Gal) activity during senescence. Using X-Gal as a substrate, the β-galactosidase staining kit generates a deep blue product under the catalysis of senescence-specific β-galactosidase. Cells expressing β-galactosidase that turn blue are easily observed under a light microscope, and their anti-senescence ability is assessed by calculating the staining positivity rate.

[0055] The cell line used was human skin fibroblasts (HSF) (Shanghai Cell Bank, Chinese Academy of Sciences). The testing conditions were: incubator temperature 37±1℃, humidity 90±5%, carbon dioxide 5±1%. Cells were cultured and treated according to groups, followed by testing. The testing methods are as follows: (1) Sample preparation: The sample (the asparagine mixture prepared in Examples 1-3 and Comparative Examples 1-8) was prepared into a stock solution of 50 mg / mL with PBS, and then diluted with DMEM medium (DME101500, Jetech) containing 10% v / v FBS to a sample solution with a concentration of 6.25 mg / mL. (2) Seed the cell suspension into a 24-well cell culture plate at a density of 100,000 cells / well, add 1 mL of DMEM culture medium containing 10% v / v FBS to each well, and culture for 24 h; (3) Sample feeding: Discard the original culture medium in each group. Add 1.0 mL of DMEM medium (DME101500, Jet) containing 10% v / v FBS to the blank (Control) group and hydrogen peroxide damage group respectively; add 1.0 mL of sample solution to the sample group respectively. Each group treatment is set up in 3 parallel experiments. (4) Damage: Except for the Control group, each group was given 0.5 mL of H2O2 (88597-100mL-F, Sigma) solution with a concentration of 3000 μmol / L. The Control group was given 0.5 mL of DMEM medium with 10% FBS. Each group was cultured for another 24 h. (5) β-galactosidase assay: Follow the instructions for the test kit (G1580, Solarbio) and calculate the staining positivity rate (%): Staining positivity rate (%) = (Number of stained cells / Total number of cells) × 100%; The lower the staining positivity rate, the stronger the anti-cellular aging ability of the asparagine mixture.

[0056] The results are averaged, as shown in Table 5. The data from Examples 1-3 show that the asparagine mixture prepared by the present invention has a strong anti-cellular aging ability. However, considering the data from Example 1 and Comparative Examples 1-8, the anti-cellular aging ability of Comparative Examples 1-8 is poor. This indicates that during the preparation of the asparagine mixture, the molecular weight cutoff of the ultrafiltration membrane, the enzymatic hydrolysis step, the alcohol precipitation step, and the raw material pretreatment step all affect the anti-cellular aging ability of the asparagine mixture. Among these, the molecular weight cutoff of the ultrafiltration membrane has the most significant effect.

[0057] Table 5. Results of HSF cell staining positivity rate Group Staining positivity rate (%) Example 1 20.41 Example 2 27.85 Example 3 23.09 Comparative Example 1 43.68 Comparative Example 2 40.42 Comparative Example 3 53.03 Comparative Example 4 51.27 Comparative Example 5 48.52 Comparative Example 6 55.16 Comparative Example 7 66.48 Comparative Example 8 62.52 Control group 8.59 Hydrogen peroxide damage group 86.43 Test Example 4: The promoting effect of aspartic peptide mixture on type I procollagen in human skin fibroblasts (HSF) This test case investigates the promoting effect of an aspartic peptide mixture on type I collagen in cells. Dermal fibroblasts synthesize type I procollagen intracellularly, which is then secreted extracellularly. Under the action of terminal procollagen peptidase, the telopeptides separate and polymerize to form collagen fibers. Type I procollagen levels can indirectly reflect type I collagen levels. Human dermal fibroblasts can serve as a cell model for studying the enhancement of type I collagen content. By measuring the upregulation rate of type I procollagen content after administration of the test substance, the efficacy of the test substance in promoting collagen synthesis can be evaluated.

[0058] The cell line used was human skin fibroblasts (HSF) (Shanghai Cell Bank, Chinese Academy of Sciences). The testing conditions were: incubator temperature 37±1℃, humidity 90±5%, carbon dioxide 5±1%. Cells were cultured and treated according to groups, followed by testing. The testing methods are as follows: (2) Sample preparation: The samples (the asparagine mixture prepared in Examples 1-3 and Comparative Examples 1-8) were prepared into a stock solution of 50 mg / mL with PBS, and then diluted with DMEM medium (DME101500, Jetech) containing 5% v / v FBS to a sample solution with a concentration of 6.25 mg / mL; the positive control TGF-β1 (HY-P70543, MCE) was prepared into a concentration of 100 ng / mL with DMEM medium (DME101500, Jetech) containing 5% v / v FBS. (2) The cell suspension was seeded into a 96-well cell culture plate at a density of 10,000 cells / well. 100 μL of DMEM culture medium containing 10% v / v FBS was added to each well and cultured for 24 h. (3) Sample feeding: Discard the original culture medium of each group. Add 200 μL of DMEM medium (DME101500, Jetech) containing 5% v / v FBS to the blank control group; add 200 μL of TGF-β1 solution with a concentration of 100 ng / mL to the TGF-β1 group; add 200 μL of sample solution to each sample group; continue to culture for 24 h in each group; set up 3 parallel experiments for each group treatment; (5) Testing: After the culture is completed, collect the cell supernatant and perform the test according to the instructions of the Human PCⅠ (Procollagen Ⅰ) ELISA Kit (E-EL-H0181, Elabscience); Upregulation rate of type I procollagen content (%) = (Procollagen I content in sample group or TGF-β1 group - Procollagen I content in blank control group) / Procollagen I content in blank control group × 100%; The higher the upregulation rate of type I procollagen content, the better the effect of the asparagine mixture on promoting collagen synthesis.

[0059] The results are shown in Table 6. The asparagine mixtures of Examples 1-3 all exhibited good effects in promoting collagen synthesis. In particular, the upregulation rate of type I procollagen content in the asparagine mixture of Example 1 was slightly higher than that in the positive control group, indicating that the asparagine mixture of Example 1 can effectively promote collagen synthesis and delay skin aging. Comparison of the data from Example 1 and Comparative Examples 1-8 shows that the molecular weight cutoff of the ultrafiltration membrane, the enzymatic hydrolysis step, the alcohol precipitation step, and the raw material pretreatment step all affect the effect of the asparagine mixture on promoting collagen synthesis. Among these, the molecular weight cutoff of the ultrafiltration membrane has the most significant impact.

[0060] Table 6 Results of the upregulation rate of type I procollagen content in HSF cells Group Upregulation rate of type I procollagen content (%) Example 1 31.9 Example 2 25.5 Example 3 27.1 Comparative Example 1 19.0 Comparative Example 2 20.3 Comparative Example 3 14.2 Comparative Example 4 16.4 Comparative Example 5 17.7 Comparative Example 6 10.2 Comparative Example 7 8.6 Comparative Example 8 9.3 TGF-β1 group 30.5 Test Example 5 This test case identifies and characterizes the aspartic peptide mixture prepared in Example 1, and uses molecular docking to study its possible anti-aging mechanism.

[0061] 1. Structural identification and characterization: First, the peptide composition of the aspartic peptide mixture prepared in Example 1 was identified and characterized by LC-MS. Then, the bioactivity of the identified peptides was predicted using PeptideRanker, and candidate sequences with an activity score >0.5 were screened. Based on this, a second screening was performed using mass spectrometry identification parameters, ultimately obtaining high-confidence peptide sequences.

[0062] The highly active peptide sequences obtained through screening are: FF, FW, WFW, MPF, FFPF, FPF, WPF, WFL, LPF, FPL, LFP, FP, GFL, FLG, LGF, and APLF. The above sequences are represented by single-letter amino acid codes, where F represents phenylalanine (Phe), W represents tryptophan (Trp), P represents proline (Pro), L represents leucine (Leu), M represents methionine (Met), G represents glycine (Gly), and A represents alanine (Ala).

[0063] The above results indicate that the anti-aging activity of the aspartic peptide mixture provided by the present invention mainly originates from the above peptide sequences: FF, FW, WFW, MPF, FFPF, FPF, WPF, WFL, LPF, FPL, LFP, FP, GFL, FLG, LGF, APLF.

[0064] 2. Molecular docking: Molecular docking is a theoretical method that predicts the binding conformation and binding free energy of ligands and receptors by simulating intermolecular interactions. It can be applied to virtual drug screening and to elucidate the mechanism of action of drugs or molecules.

[0065] SIRT, short for Sirtuins, are class III histone deacetylases dependent on nicotinamide adenine dinucleotide (NADP). They participate in regulating many important biological processes, including maintaining genome stability, regulating metabolism, and regulating stem cells. SIRT 2 can regulate the activity of FoxO3 transcription factor in fibroblasts, reducing H2O2-mediated reactive oxygen species production and influencing cellular senescence. ERK, short for extracellular regulated protein kinases, includes ERK1 and ERK2. The ERK1 / 2 pathway plays a crucial role in combating oxidative damage and regulating aging. mTOR is a serine / threonine protein kinase that forms two structurally and functionally distinct complexes, mTORC1 and mTORC2, by binding with other proteins. mTORC1 and mTORC2 form signaling pathways with different upstream and downstream proteins to regulate cell growth, proliferation, and survival.

[0066] This invention first uses ChemDraw to construct the two-dimensional planar structure of the above peptide; then the structure is imported into Chem3D to generate a three-dimensional conformation, and the MM2 force field is used to perform preliminary geometric optimization; from the RCSB PDB database, the PDB format files of anti-aging related targets "Sirt2", "ERK1" and "mTOR" are downloaded, and molecular docking is performed using AutoDock software.

[0067] The molecular docking results of the aspartic peptide mixture with targets Sirt2, ERK1, and mTOR are as follows: Figure 1 As shown, molecular docking results indicate that the above-mentioned peptides can bind to the targets Sirt2, ERK1, and mTOR. Therefore, the aspartic peptide mixture provided by this invention may achieve its anti-aging effect through the targets Sirt2, ERK1, and mTOR and their related pathways.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A mixture of aspartic peptides, characterized in that, The asparagine peptide mixture is an active peptide mixture with a molecular weight of <5 kDa, prepared from asparagus tuber as raw material by enzymatic hydrolysis.

2. The aspartic peptide mixture as described in claim 1, characterized in that, The molecular weight of the aspartic peptide mixture is <1 kDa.

3. The asparagine mixture of claim 1, wherein the asparagine mixture comprises short peptides with the following amino acid sequences: FF, FW, WFW, MPF, FFPF, FPF, WPF, WFL, LPF, FPL, LFP, FP, GFL, FLG, LGF, APLF.

4. The aspartic peptide mixture as described in claim 1, characterized in that, The preparation method of the aspartic peptide mixture includes the following steps: S1. Crush the dried asparagus tubers to obtain asparagus powder, add pure water, stir and soak at 4-10℃ for 20-50 minutes, and treat with pulse ultrasound to obtain pretreated material; S2. Adjust the pH of the pretreated material to 7.2-8.5, add trypsin, carry out the first stirring reaction, filter with a filter cloth to obtain filter residue and the first peptide filtrate; take the filter residue, add pure water, adjust the pH to 6.2-7.5, then add papain and neutral protease, carry out the second stirring reaction, filter with a filter cloth to obtain the second peptide filtrate; combine the two peptide filtrates, inactivate the enzymes, filter by suction to obtain a clear filtrate; S3. Concentrate the clarified filtrate, precipitate it with ethanol, and centrifuge to obtain the supernatant. After removing the ethanol from the supernatant, use an ultrafiltration membrane system to retain the permeate with a molecular weight <5KDa and collect the permeate. S4. The permeate is freeze-dried under vacuum to obtain a mixture of aspartic peptides.

5. The aspartic peptide mixture as described in claim 4, characterized in that, The conditions for pulsed ultrasound treatment in step S1 are: power 300W, frequency 20~40kHz, pulse mode of 3s working and 3s intermittent, and ultrasound treatment time of 10-30min.

6. The aspartic peptide mixture as described in claim 4, characterized in that, In step S2, the amount of trypsin added is 1%-4% of the mass of asparagus powder; the temperature of the first stirring reaction is 45-55℃, the rotation speed is 300-500 r / min, and the time is 60 min; the amount of papain added is 1%-2% of the mass of asparagus powder; the amount of neutral protease added is 0.5%-1.5% of the mass of asparagus powder; the temperature of the second stirring reaction is 45-55℃, the rotation speed is 300-500 r / min, and the time is 60 min.

7. The aspartic peptide mixture as described in claim 4, characterized in that, The concentration mentioned in step S3 is vacuum concentration to 1 / 5 to 1 / 10 of the original volume; the ethanol precipitation is to slowly add 95% v / v ethanol to the concentrate while stirring at a speed of 120-150 r / min until the final ethanol concentration in the system reaches 85% v / v, and then let it stand at 4°C for 10-16 h.

8. The use of the aspartic peptide mixture according to any one of claims 1-7 in the preparation of products for delaying skin aging.

9. The application as described in claim 8, characterized in that, The effective concentration of the asparagine mixture in the product is not less than 1.0 mg / mL.

10. A cosmetic product for delaying skin aging, characterized in that, A mixture containing the asparagine of any one of claims 1-7.

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