Anti-aging composition and use thereof

Abstract

The application discloses an anti-aging composition and application thereof. The anti-aging composition is prepared by compounding dimethylsilyl hyaluronate, radix pseudoginseng cyclic peptide B and alfalfa extract. The alfalfa extract is obtained by adopting pulse ultrasonic assisted subcritical extraction technology, and can play a better anti-aging effect. The experiment proves that the composition can significantly improve the contents of retinoic acid binding receptor RAR alpha and RXR gamma protein and type I collagen protein, inhibit cell aging, and achieve an anti-aging effect similar to retinol. Compared with retinol, the composition is mild and non-irritating, can significantly improve the preservative efficiency of a product in skin care, and can reduce the amount of preservatives.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cosmetics, and particularly relates to an anti-aging composition and application thereof. BACKGROUND

[0002] Skin aging is a complex biological process involving multiple intrinsic and extrinsic factors. Essentially, it is driven by the impairment, decline, and death of skin cells. These changes not only affect the appearance of the skin, but also its physiological functions, such as regenerative capacity and barrier effect. Fibroblasts play a central role in the structure and function of the skin, as they are responsible for the synthesis and secretion of collagen, elastin, and extracellular matrix, which helps to repair damaged skin and reduces the impact of age-related aging on the skin. Fibroblast aging is a gradual accumulation of cellular aging, including double-stranded DNA breaks, DNA oxidative damage, chromosomal abnormalities, epigenetic changes, and telomere shortening.

[0003] Retinoic acid and its derivatives exert their effects through nuclear receptors, including retinoic acid receptors (RARα, β, γ) and retinoid X receptors (RXRα, β, γ), both of which form heterodimers that bind to DNA, activate RAR and RXR-related genes, and upregulate collagen gene expression, counteracting the loss of extracellular matrix caused by aging.

[0004] Type I collagen is the most abundant collagen in the human body, accounting for about 90% of total collagen, and is the main component of the dermis, working together with elastic fibers, hyaluronic acid, and others to maintain skin elasticity and tensile strength, reducing wrinkle formation. Age-related beta-galactosidase (SA-β-gal) is a lysosomal enzyme commonly used to mark cell aging. When cells age, the number and volume of lysosomes increase, leading to upregulation of SA-β-gal activity. The number of senescent cells can be identified by beta-galactosidase staining. Hyaluronic acid is widely used in skincare products due to its moisturizing properties, but there is little research on its anti-aging properties. Therefore, the present application explores whether the combination of hyaluronic acid, radix pseudoginseng ring peptide, and alfalfa extract can also improve the aging and morphology of human skin fibroblasts, increasing the anti-aging properties of hyaluronic acid on the basis of moisturizing, and filling the application gap of hyaluronic acid in the anti-aging field. SUMMARY

[0005] To solve the above problems, the present application provides an anti-aging composition and application thereof. The present application combines dimethylsilanol hyaluronate (abbreviated as DSHA), radix pseudoginseng ring peptide B, and alfalfa extract to form an anti-aging composition; experiments have verified that the composition has good anti-aging efficacy, and the composition is mild and non-irritating, can significantly improve the preservative efficacy of skincare products, and can reduce the amount of preservatives used.

[0006] In order to achieve the above technical purposes, the technical scheme provided by the present application is as follows:

[0007] In a first aspect of the present application, an anti-aging composition is provided, and the effective components of the anti-aging composition are as follows in terms of mass fraction: 0.01-0.1 parts of DSHA, 0.05-0.2 parts of radix pseudostellariae cyclic peptide B, and 1.0-15.0 parts of alfalfa extract.

[0008] Preferably, the effective components of the anti-aging composition are as follows in terms of mass fraction: 0.03-0.08 parts of DSHA, 0.08-0.15 parts of radix pseudostellariae cyclic peptide B, and 5.0-10.0 parts of alfalfa extract.

[0009] The alfalfa extract is extracted by a pulsed ultrasonic assisted subcritical extraction method, and the extraction method is as follows: dry alfalfa stems and leaves are crushed, water is added, and pulsed ultrasonic extraction is performed at room temperature; then water is continuously added, and subcritical extraction is performed under the conditions of a temperature of 120-140 DEG C and a pressure of 0.8-1.2 MPa; the filtrate is concentrated under reduced pressure to 4-6 times the mass of the alfalfa powder; 1-1.5 times the amount of glycerol is added to the concentrated solution; the mixture is stirred and uniformly mixed; and then the alfalfa extract is obtained after filtration, wherein the content of flavonoid components in the extract is 0.6-1.0 mg / mL, and the content of polyphenol components in the extract is 1.0-1.8 mg / mL.

[0010] The preparation method is as follows: DSHA, radix pseudostellariae cyclic peptide B and alfalfa extract are mixed and dissolved in purified water to make up 100 parts.

[0011] In a second aspect of the present application, the anti-aging composition is applied to the preparation of an anti-aging skin care product. Through experiments, it has been verified that the above-mentioned composition can promote the expression of retinoic acid binding receptor RARα and RXRγ protein, promote the generation of type I collagen, and inhibit cell aging, so as to achieve the anti-aging effect. It has also been verified through experiments that the composition can significantly improve the preservative efficiency of the product and reduce the amount of preservatives in the skin care product.

[0012] Further, the amount of the anti-aging composition added is 1-20% (w / w) based on the total mass of the anti-aging skin care product.

[0013] The anti-aging skin care product can also contain other raw material components allowed to be added in the field of skin care products, including but not limited to emulsifiers, emollients, moisturizers, cooling agents, skin conditioners, thickening agents and preservatives, etc.

[0014] Meanwhile, the above-mentioned raw material components can also be reasonably added to prepare different cosmetic dosage forms such as essence water, essence milk, essence cream, etc., which are not specifically limited herein.

[0015] The present application has the following beneficial technical effects:

[0016] (1) The alfalfa extract of the present application is obtained by using pulse ultrasonic assisted subcritical extraction technology, compared with the traditional water extraction and alcohol precipitation method, the heat-sensitive components are protected, the decomposition of heat-sensitive components in the extraction process is prevented, and better anti-aging effect is achieved; water is used as the extraction solvent, and no organic solvent is used, which is more green and environmentally friendly.

[0017] (2) The anti-aging composition obtained by the above technical solution can significantly increase the content of retinoic acid binding receptor RAR alpha and RXR gamma protein and type I collagen, inhibit cell aging, and achieve anti-aging effect similar to retinol, but compared with retinol, it is mild and non-irritating.

[0018] (3) The anti-aging composition prepared by the above technical solution has good preservative performance in skin care products, and can reduce or replace the use of traditional preservatives. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Gradient dilution diagram of bacteria;

[0020] Figure 2 Gradient dilution diagram of fungi. DETAILED DESCRIPTION

[0021] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present application. Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application pertains.

[0022] It should be noted that the terms used herein are only for the purpose of describing specific embodiments, and are not intended to limit the exemplary embodiments according to the present application. As used herein, the singular form is intended to include the plural form unless the context clearly indicates otherwise, and it should be understood that when the terms "comprise" and / or "include" are used in the specification, there is a feature, step, operation, device, component and / or combination thereof.

[0023] The present application will be further described with reference to specific examples, which are only intended to explain the present application and do not limit its content. If the specific conditions of the experiments are not specified in the examples, they are usually carried out under conventional conditions or according to the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples, if not specified, can be obtained from commercial channels.

[0024] The application is further explained by the following examples, which do not limit the application. It should be understood that these examples are merely for the purpose of illustration of the application and are not intended to limit the scope of the present application. The test methods in the following examples, unless otherwise stated, were carried out under conventional conditions. It should be noted that the DSHA in the examples and comparative examples of the present application is from Shandong Furuida Biological Co., Ltd., and the trade name is Silanized Hyaluronic Acid, and the INCI name is Dimethylsilanol Hyaluronate; the Radix Pseudoginseng Cyclopeptide B in the examples and comparative examples is purchased from Shanghai Xiaowei Biological Technology Co., Ltd., and the trade name is PortaVita TM HB Radix Pseudoginseng Cyclopeptide; the hyaluronic acid (3w-5w Da) in the comparative examples is purchased from Shandong Jiaofeng Furuida Biological Co., Ltd.; the retinol in the comparative examples is purchased from Nanjing Anyike Chemical Co., Ltd.

[0025] In the examples and comparative examples of the present application, the method for obtaining the alfalfa extract is as follows:

[0026] 1. Water extraction and alcohol precipitation method: dry alfalfa is crushed and passed through a 60-mesh sieve, and water reflux extraction is carried out twice, with a solid-liquid ratio of 1:10, and each time for 2 h. The combined extract is concentrated under reduced pressure to about 3 times the amount of medicinal materials. An appropriate amount of 95% ethanol solution is added to the concentrated solution to a content of 70% (w / w), stirred for 10 min, and refrigerated (4±2℃) for 12 h or more. The filtrate is recovered under reduced pressure using a sterilization filter plate and a PP membrane, and the ethanol is recovered at 60℃ under reduced pressure. The filtrate is concentrated to no alcohol taste, and further concentrated to about 3 times the amount of medicinal materials. Glycerol is added to the concentrated solution at a concentration of 1.8 times the mass of the concentrated solution, and the mixture is stirred and filtered to obtain the alfalfa extract. The concentration of the alfalfa extract is 0.1227 g / g based on the amount of crude drug.

[0027] 2. Pulsed ultrasonic assisted subcritical extraction method:

[0028] (1) Dry alfalfa stems and leaves are crushed and passed through a 60-mesh sieve, and water is added for pulsed ultrasonic extraction (working 2 s / intermittent 1 s) twice, with a solid-liquid ratio of 1:10, an ultrasonic power of 400 W, a temperature of 25℃, and each time for 30 min. After ultrasonic extraction, the solution is used for the next step;

[0029] (2) Place the liquid prepared in step (1) into the extractor and continue to add water. The amount of water added is based on a mass ratio of alfalfa powder to water of 1:20 (the amount of water used for pulsed ultrasound is small, while the amount of water used for subcritical fluid extraction is large, so water needs to be added). The extraction conditions are: temperature of 140℃, pressure of 1.0MPa, and time of 40min. Filter the extract and concentrate the filtrate under reduced pressure to about 5% of the mass of alfalfa powder. Add 1.2 times the amount of glycerol to the concentrate, stir and mix evenly, and filter to obtain alfalfa extract. Quantitative analysis of the alfalfa extract is performed on the content of flavonoids and polyphenols. The content of flavonoids in the extract is 0.7241mg / mL, and the content of polyphenols in the extract is 1.3522mg / mL.

[0030] Examples 1-3:

[0031] The specific formulations for Examples 1-3 and Comparative Examples 1-11 are shown in Table 1 below, calculated by mass parts. The preparation method was as follows: DSHA, Codonopsis pilosula cyclic peptide B, alfalfa extract, etc., were mixed according to the amounts used in each example and comparative example, and dissolved in purified water according to the formulations in Table 1. Since retinol (VA) is insoluble in water, DMSO was used for dispersion and dissolution during in vitro evaluation.

[0032] Table 1. List of specific examples and comparative components demonstrating anti-aging effects.

[0033]

[0034] Note: Alfalfa extract 1 was prepared by pulsed ultrasound-assisted subcritical extraction technology, and alfalfa extract 2 was prepared by water extraction and alcohol precipitation.

[0035] The essence water prepared according to the formula in Table 2 with the addition of DSHA, Codonopsis pilosula cyclic peptide B and alfalfa extract is Example 4. Comparative Example 12 is an essence water using a classic preservative system, and Comparative Example 13 is an essence water without the anti-aging composition compared to Example 4. Note: All raw materials in Table 2 are sterile, and the preparation of Example 4, Comparative Example 12, and Comparative Example 13 were all completed under sterile conditions.

[0036] Table 2. List of specific examples and comparative components for evaluating the antiseptic efficacy of essence water.

[0037]

[0038]

[0039] Preparation method of essence water:

[0040] S1: Add the raw materials in phase A separately, heat to 80-85℃ and stir to dissolve. After complete dissolution, add phase B. After complete dissolution, start cooling.

[0041] S2: When the temperature drops to 45℃, add phase C, stir to dissolve and disperse evenly to obtain the essence water product.

[0042] Experimental Example 1: Stimulus Testing Experiment

[0043] In accordance with SN / T 2329-2009 "Eye Irritation of Cosmetics / Chicken Embryo Villi Allantoic Membrane Test", the product was subjected to an eye irritation test, which is also known as a safety test.

[0044] Experimental method: Fertilized chicken embryos under 7 days old were incubated for 9 days in an incubator at 37.6±0.5℃ and 50%~70% humidity.

[0045] CAM preparation: Candling is performed to check the egg. The location of the air cell is marked on the eggshell surface. The marked portion of the eggshell is peeled off with tweezers to expose the white egg membrane. Care should be taken not to damage the integrity of the egg membrane. 0.5 mL of 0.9% NaCl solution is added to fully moisten the egg membrane. The surface liquid is gently absorbed with a paper towel, and the inner membrane is carefully removed with tweezers, ensuring that the vascular membrane is not damaged.

[0046] Reaction time method: 0.3 mL of the transparent test substance was evenly added or spread onto the CAM surface. After 3 min of action, the test substance was washed away with 0.9% NaCl solution. The degree of change of each toxic effect of CAM was observed, and the time of occurrence of each toxic effect within 5 min of action was recorded. Six chicken embryos were set up for each test substance, and one chicken embryo was set up for the negative control (physiological saline) and one for the positive control (1% SDS). The scoring criteria and phenomena are shown in Table 3.

[0047] Table 3 Scoring Criteria and Phenomena

[0048]

[0049]

[0050] Stimulus rating method: The reaction time method is used for the experiment. The stimulus rating (IS) is calculated using the following formula, and the result is rounded to two decimal places:

[0051]

[0052] In the formula:

[0053] secH (bleeding time) ----- The average time for the onset of bleeding observed on the CAM membrane, measured in seconds (s);

[0054] secL (vascular dissolution time) ----- The average time for vascular dissolution to begin as observed on the CAM membrane, measured in seconds (s);

[0055] secC (clotting time) ----- The average time for clotting to begin as observed on a CAM membrane, measured in seconds (s).

[0056] The eye irritation of the test substances was classified according to the IS values ​​in Table 4.

[0057] Table 4 Evaluation of Stimulus Rating Method Results

[0058] Irritation score Irritation classification IS < 1 Non-irritating 1 < IS < 5 Mildly irritating 5 < IS < 10 Moderately irritating IS > 10 Strongly irritating / corrosive

[0059] As shown in Table 5, the anti-aging compositions of Examples 1-3 and Comparative Examples 1-6 and 8-11 showed no irritation in the chicken embryo chorioallantoic membrane test, indicating that one, two, or three combinations of DSHA, Codonopsis pilosula cyclic peptide B, and alfalfa extract are safe, non-toxic, and have low irritation. The IS value of Comparative Example 7 was 8.42, indicating moderate irritation (Comparative Example 7 was prepared using olive oil with a retinol content of 0.05%). This demonstrates that the combined use of DSHA, Codonopsis pilosula cyclic peptide B, and alfalfa extract reduces irritation compared to retinol.

[0060] Table 5 Statistical Table of Stimulation Test Results

[0061]

[0062]

[0063] Experiment 2: In vitro experiment to promote the activity of retinoic acid binding receptors RAR-α and RXR-γ

[0064] This experiment used H2O2 to treat human skin fibroblasts (HSF) (purchased from Guangdong Boxi Biotechnology Co., Ltd., batch number: 20081902, hereinafter the same). The expression levels of RAR-α and RXR-γ in the samples were detected by ELISA to evaluate the ability of the test substance to promote the expression of retinoic acid binding receptors RAR-α and RXR-γ. According to the test protocol in Table 6, when the fibroblast deposition rate in the 12-well plate reached 40%–60%, HSF was treated with 400 μM H2O2 for 24 h. Drug administration was performed according to the test groups, with 2 mL of drug added to each well, and each group had 3 replicates. The blank group and model group were added with 2 mL of 10% FBS complete medium (Lanzhou Rongye), while the sample group was added with 2 mL of medium containing the corresponding concentration of the test substance and 10% FBS complete medium. After drug administration, the 12-well plates were placed in an incubator (37℃, 5% CO2) for 24 h. Detection was performed according to the ELISA kit (Abcam, hereinafter the same). The test results are shown in Tables 7 and 8.

[0065] Table 6 Test Plan

[0066]

[0067] Note: Sample concentration 2 mg / mL (diluted with deionized water; Comparative Example 7 was prepared by dissolving in DMSO to a 0.05% retinol solution, and the sample concentration was 2 mg / mL).

[0068] Table 7 Summary of RAR-α Expression Level Detection Results

[0069]

[0070] Note: ## indicates p < 0.01 compared to the control group; ** indicates p < 0.01 compared to the model group; * indicates p < 0.01 compared to the model group. <p<0.05。

[0071] As shown in Table 7, Comparative Examples 3 and 10 indicate that alfalfa extract alone does not promote RAR-α expression. Comparative Examples 1 and 8 show that DSHA alone and sodium hyaluronate (3w-5w Da) have a certain promoting effect on RAR-α receptor expression. Comparative Example 9 indicates that the synergistic effect of sodium hyaluronate (3w-5w Da), codonopsis pilosula cyclic peptide B, and alfalfa extract 1 is not significant. Comparative Example 11 indicates that the synergistic effect of DSHA, codonopsis pilosula cyclic peptide B, and alfalfa extract 2 is not as significant as in the examples. Examples 1-3 all significantly upregulated RAR-α content, indicating that DSHA, codonopsis pilosula cyclic peptide B, and alfalfa extract 1 have a significant synergistic promoting effect. Meanwhile, Example 2 is comparable in efficacy to Comparative Example 7 (retinol alone).

[0072] Table 8 Summary of RXR-γ Expression Level Detection Results

[0073]

[0074] Note: ## indicates p < 0.01 compared to the control group; ** indicates p < 0.01 compared to the negative control group; * indicates p < 0.01 compared to the negative control group. <p<0.05。

[0075] Table 8 shows that Comparative Examples 1, 3, 8, and 10 demonstrate that DSHA alone, sodium hyaluronate (3w-5w Da), and alfalfa extract have a certain promoting effect on RXR-γ receptor expression. Examples 1-3 all significantly upregulated RXR-γ gene expression. Meanwhile, Example 2 and Comparative Example 7 (retinol alone) showed comparable efficacy, indicating that DSHA, ginseng cyclic peptide B, and alfalfa extract 1 have a significant synergistic promoting effect. The effect of Comparative Example 9 was lower than that of Examples 1-3, indicating that sodium hyaluronate (3w-5w Da), ginseng cyclic peptide B, and alfalfa extract 1 did not have a significant synergistic promoting effect. Compared with Comparative Example 11, the synergistic effect of alfalfa extract 1 with DAHA and ginseng cyclic peptide B was superior to that of alfalfa extract 2 alone.

[0076] Experiment Example 3: In vitro assay to determine the percentage of senescent cells after β-galactosidase staining

[0077] The effect of the anti-aging composition on cellular senescence levels was detected using the β-galactosidase staining method. This method included the following steps: According to the test groups in Table 9, when the HSF plating rate in the 6-well plate reached 40%–60%, the plate was stimulated with 300 μM H2O2 for 2 hours. Drug administration was performed according to the test groups, with 2 mL of sample added to each well, and each group had 3 replicates. For the blank control group and negative control group, 2 mL of DMEM culture medium (Thermo Fisher Scientific, hereinafter the same) containing 5% serum was added to each well. For the positive control group, 2 mL of DMEM culture medium containing 50 ng / ml TGF-β1 and 5% serum was added to each well. For the sample group, 2 mL of DMEM culture medium containing the corresponding concentration of the test substance and 5% serum was added to each well. After drug administration, the 6-well plate was placed in an incubator and incubated for 22 hours. Cells were digested with 0.25% trypsin (Thermo Fisher Scientific), seeded into 6-well plates, and incubated for 24 hours. The cell culture medium was aspirated, and the cells were washed once with PBS. 1 mL of β-galactosidase staining and fixative was added, and the plates were fixed at room temperature for 15 minutes. The cell fixative was aspirated, and the cells were washed three times with PBS. PBS was aspirated, and 1 mL of staining working solution was added to each well. The plates were incubated overnight at 37°C. The plates were sealed with sealing film to prevent evaporation. The inhibition rates were observed and photographed under a light microscope. The inhibition rate results are shown in Table 10. The formula for calculating the inhibition rate is as follows:

[0078]

[0079] Table 9 Test Plan

[0080]

[0081]

[0082] Note: Sample concentration 2 mg / mL (diluted with deionized water; Comparative Example 7 was prepared by dissolving in DMSO to a 0.05% retinol solution, and the sample concentration was 2 mg / mL).

[0083] Table 10 Results of Fibroblast Senescence Level Test

[0084]

[0085] Note: ## indicates p < 0.01 compared to the control group; ** indicates p < 0.01 compared to the control group; * indicates p < 0.01 compared to the control group. <p<0.05。

[0086] As shown in Table 10, Comparative Examples 1-11 indicate that DSHA, sodium hyaluronate (3w-5w Da), codonopsis pilosula cyclic peptide B, and alfalfa extract, used alone or in combination, have a certain inhibitory effect on fibroblast senescence. Retinol alone has no significant effect on improving cell senescence. Examples 1-3 all significantly reduced the percentage of β-galactosidase-positive cells after staining, and this reduction was positively correlated with concentration, indicating that the synergistic effect of DSHA, codonopsis pilosula cyclic peptide B, and alfalfa extract 1 can significantly inhibit fibroblast senescence. The inhibitory abilities of Comparative Examples 9 and 11 were lower than those of Examples 1-3, indicating that the synergistic effect among sodium hyaluronate (3w-5w Da), codonopsis pilosula cyclic peptide B, and alfalfa extract was not significant, and the synergistic effect of alfalfa extract 1 was better than that of alfalfa extract 2.

[0087] Experiment 4: Experiment on promoting type I collagen synthesis

[0088] This experiment used UVA irradiation of HSF to detect changes in the content of type I collagen (Collagen I) in the samples. According to the test protocol in Table 11, when the fibroblast deposition rate in the 24-well plate reached 40%–60%, the samples were grouped and administered the drug. 2 mL of sample was added to each well, with 3 replicates per group. For the blank control group and negative control group, 2 mL of DMEM culture medium containing 5% serum was added to each well. For the positive control group, 2 mL of DMEM culture medium containing 100 ng / ml TGF-β1 and 5% serum was added to each well. For the sample group, 2 mL of DMEM culture medium containing the corresponding concentration of the test substance and 5% serum was added to each well. After drug administration, the 6-well plates were placed in an incubator (37℃, 5% CO2) for 24 h. Based on the experimental groups, groups requiring UVA irradiation were subjected to 30 J / cm² irradiation. 2 The sample was irradiated with UVA for 26 minutes and then incubated in an incubator (37℃, 5% CO2) for 24 hours. Detection was performed according to the ELISA kit instructions. The test results are shown in Table 12.

[0089] Table 11 Test Plan

[0090]

[0091] Note: Sample concentration 2 mg / mL (diluted with deionized water; Comparative Example 7 was prepared by dissolving in DMSO to a 0.05% retinol solution, and the sample concentration was 2 mg / mL).

[0092] Table 12 Summary of Collagen I Content Results

[0093]

[0094] Note: ## indicates p < 0.01 compared to the control group; ** indicates p < 0.01 compared to the negative control group; * indicates p < 0.01 compared to the negative control group. <p<0.05。

[0095] Table 12 shows that Comparative Examples 1, 2, and 8 indicate that the use of DSHA, sodium hyaluronate (3w-5w Da), and Codonopsis pilosula cyclic peptide B alone has a certain promoting effect on the production of type I collagen. Comparative Examples 3 and 11, using only alfalfa extract, showed no significant promoting effect. Examples 1-3 all significantly promoted the production of type I collagen, with Example 2 showing the most significant effect. This indicates that the combined use of DSHA, Codonopsis pilosula cyclic peptide B, and alfalfa extract 1 can significantly promote the production of type I collagen and has a synergistic effect.

[0096] Experiment Example 5: Corrosion Protection Performance Evaluation Experiment

[0097] Experimental strains:

[0098] 1. Bacteria: Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, all of which were third-generation cultures;

[0099] 2. Fungi: Aspergillus niger and Candida albicans, both of which are third-generation cultures.

[0100] Experimental steps:

[0101] 1. Preparation of bacterial suspensions: The cryopreserved strains of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa were inoculated separately into tryptic soy agar medium and incubated at 32°C for 48 hours. The suspensions were then serially diluted with 0.85% physiological saline, with 2 ml of each dilution injected into two sterile Petri dishes (1 ml per dish). The Petri dishes were then plated with tryptic soy agar medium and incubated at 32°C for 48 hours. Bacterial counts were then performed. 10... 8 Mix equal volumes of bacterial suspensions of similar magnitude for later use.

[0102] 2. Preparation of Candida albicans suspension:

[0103] The cryopreserved strain of *Candida albicans* was inoculated into Sabouraud dextrose liquid medium and incubated at 28°C for 5 days. It was then serially diluted with 0.85% physiological saline, with 2 ml of each dilution injected into two sterile Petri dishes (1 ml per dish). The plates were then poured onto Sabouraud dextrose agar and incubated at 28°C for 5 days. The bacteria were then counted. 10... 6 Prepare orders of magnitude bacterial suspensions for later use.

[0104] 3. Preparation of Aspergillus niger spore suspension:

[0105] The above-mentioned cryopreserved spore suspension of *Aspergillus niger* was inoculated onto Sabouraud dextrose agar slants and incubated at 28°C for 5–7 days. The mycelial growth was then washed with 25 ml of 0.85% physiological saline containing 0.05% Tween 80 to prepare a spore suspension. This suspension was serially diluted with 0.85% physiological saline containing 0.05% Tween 80, with 2 ml of each dilution injected into two sterile Petri dishes (1 ml per dish). The dishes were then plated on Sabouraud dextrose agar and incubated at 28°C for 72 hours. Spore counts were then performed. 10... 6 Prepare a quantity-scale spore suspension for later use.

[0106] 4. Gradient dilution plot

[0107] 1) Bacterial gradient dilution diagram as shown Figure 1 As shown.

[0108] 2) The gradient dilution diagram of fungi is shown below. Figure 2 As shown.

[0109] 5. Sample preparation: Weigh 3 portions of each sample, each weighing 120g, into a sterile conical flask for later use.

[0110] 6. Sample inoculation: Take the three weighed samples above and inoculate them with 1.2 ml of bacterial mixed suspension, 1.2 ml of Candida albicans suspension and 1.2 ml of Aspergillus niger spore suspension respectively. Label them, mix them evenly, and store the samples at room temperature.

[0111] 7. Sample testing: The prepared samples were taken on days 0, 1, 3, 7, 14, 21, and 28, and tested according to the cosmetic microbiology testing procedures. The number of colonies in the samples was recorded to determine the preservative efficacy of the cosmetics.

[0112] 8. Results of bacterial suspension counting:

[0113] The bacterial suspension counting results are shown in Table 13. The initial inoculum concentration was 1.4 × 10⁻⁶. 6 CFU / g (initial log value 6.15).

[0114] Table 13 Results of bacterial suspension counts

[0115]

[0116]

[0117] The results of the Candida albicans suspension counting are shown in Table 14. The initial inoculum concentration of the sample was 1.5 × 10⁻⁶. 4 CFU / g (initial logarithm 4.18).

[0118] Table 14 Results of Candida albicans suspension counting

[0119] Strain name Count result C. albicans 1.5 x 10 6 CFU / ml

[0120] The results of the Aspergillus niger spore suspension count are shown in Table 15. The initial inoculum concentration of the sample was 1.2 × 10⁻⁶. 4 CFU / g (initial log value 4.08).

[0121] Table 15 Results of Aspergillus niger spore suspension counting

[0122] Strain name Count result A. niger 1.2 x 10 6 CFU / ml

[0123] The preservative effect of cosmetics is evaluated using the logarithmic reduction value. The calculation method for this indicator is shown in Equation 1.

[0124] R X =lgN0-lgN X Equation 1

[0125] In the formula:

[0126] N0—The initial concentration of the bacterial solution in the sample;

[0127] N X —The number of bacterial colonies in the sample at different testing times.

[0128] The evaluation criteria for the preservative effect of cosmetics are shown in Table 16, and the sample counting results are shown in Table 17.

[0129] Table 16 Evaluation Criteria for the Preservative Effect of Cosmetics

[0130]

[0131] Table 17 Sample Count Results

[0132]

[0133]

[0134] Comparative Example 12 is a classic preservative system. According to the results in Table 17, both Example 4 and Comparative Example 12 passed the preservative efficacy standard test, and Example 4 showed superior preservative efficacy against bacteria and Aspergillus niger compared to Comparative Example 12. Comparative Example 13 failed the preservative efficacy evaluation experiment. This indicates that the combined use of DSHA, Codonopsis pilosula cyclic peptide, and alfalfa extract 1 can achieve a certain synergistic effect in promoting preservative efficacy, reducing the amount of preservatives in the formulation, and thus reducing skin irritation from preservatives.

[0135] It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the given examples, those skilled in the art can modify or make equivalent substitutions to the technical solutions of the present invention as needed, without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. An anti-aging composition, characterized in that, The effective components of the anti-aging composition are DSHA 0.01-0.1 parts, radix pseudostellariae cyclic peptide B 0.05-0.2 parts, and alfalfa extract 1.0-15.0 parts by mass; the DSHA is dimethylsilyl hyaluronate; the alfalfa extract is prepared by crushing dried alfalfa stems and leaves, adding water and performing pulse ultrasonic extraction at room temperature, continuously adding water and performing subcritical extraction at a temperature of 120-140 DEG C and a pressure of 0.8-1.2 MPa, concentrating the filtrate under reduced pressure to 4-6 times the mass of the alfalfa powder, adding 1-1.5 times the concentrated solution of glycerol, stirring and mixing uniformly, and filtering to obtain the alfalfa extract; the content of flavone components in the alfalfa extract is 0.6-1.0 mg / mL, and the content of polyphenol components in the alfalfa extract is 1.0-1.8 mg / mL.

2. The anti-aging composition according to claim 1, wherein the anti-aging composition is a cosmetic composition. The effective components of the anti-aging composition are DSHA 0.03-0.08 parts, radix pseudostellariae cyclic peptide B 0.08-0.15 parts, and alfalfa extract 5.0-10.0 parts by mass.

3. The anti-aging composition according to claim 1 or 2, characterized in that, The DSHA, radix pseudostellariae cyclic peptide B, and alfalfa extract are mixed and dissolved in purified water to make up 100 parts.

4. Use of the anti-aging composition of claim 1 or 2 in the preparation of an anti-aging skin care product.

5. An anti-aging skin care product, characterized by, The anti-aging composition of claim 1 or 2 is used as the effective component.

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