Anti-aging composition and application thereof

Abstract

The invention discloses an anti-aging composition and application thereof. According to the anti-aging composition disclosed by the invention, the anti-aging composition is prepared by compounding dimethyl silanol hyaluronate, radix pseudostellariae cyclic peptide B and a medicago sativa extract. The medicago sativa extract disclosed by the invention is obtained by adopting a pulsed ultrasound-assisted subcritical extraction technology, and can play a better anti-aging effect. Experiments verify that the composition can significantly increase the content of retinoic acid binding receptor RAR alpha and RXR gamma proteins and type I collagen, inhibit cell aging and achieve an anti-aging effect similar to that of retinol, but compared with retinol, the composition is mild and non-irritant, can significantly improve the preservative efficacy of a product when being used in skin care products, and can reduce the dosage of preservatives.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and particularly relates to an anti-aging composition and its application. Background Art

[0002] Skin aging is a complex biological process involving multiple internal and external factors. Essentially, it is driven by the damage, 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. They are responsible for synthesizing and secreting collagen, elastin, and extracellular matrix, which helps repair damaged skin and reduce the impact of aging on the skin. Fibroblast aging is a gradually accumulating cellular aging, including double-stranded DNA breaks, DNA oxidative damage, chromosomal abnormalities, epigenetic changes, telomere shortening, etc.

[0003] Retinoic acid and its derivatives act through nuclear receptors, including retinoic acid receptors (RARα, β, γ) and retinoid X receptors (RXRα, β, γ). The two form heterodimers that bind to DNA and activate RAR- and RXR-related genes, which can upregulate collagen gene expression and counteract 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 the total collagen. It is the main component of the dermis layer and, together with elastic fibers, hyaluronic acid, etc., maintains the elasticity and tensile strength of the skin and reduces the formation of wrinkles. Senescence-associated β-galactosidase (SA-β-gal) is a lysosomal enzyme commonly used to label cellular senescence. When cells age, the number and volume of lysosomes increase, leading to an upregulation of SA-β-gal activity. The number of senescent cells can be identified by β-galactosidase staining. Hyaluronic acid is widely used in skin care products due to its moisturizing properties, but little research has been done on its anti-aging properties. In this regard, the present invention combines and synergistically applies hyaluronic acid, pseudostellaria cyclic peptide, and alfalfa extract to explore whether it can also play a role in improving the aging and morphology of human skin fibroblasts, increasing the anti-aging performance of hyaluronic acid on the basis of moisturization, and filling the application gap of hyaluronic acid in the anti-aging field. Summary of the Invention

[0005] In view of the above problems, the present invention provides an anti-aging composition and its application. The present invention prepares an anti-aging composition by compounding dimethylsilanol hyaluronate (abbreviated as DSHA), pseudostellaria cyclic peptide B, and alfalfa extract. Through experimental verification, this composition has good anti-aging effects. At the same time, this composition is mild and non-irritating, can significantly improve the anti-corrosion efficiency of products when used in skin care products, and can reduce the amount of preservatives used.

[0006] To achieve the above technical objectives, the technical solution provided by the present invention is as follows:

[0007] In the first aspect of the present invention, an anti-aging composition is provided. The active ingredients of the anti-aging composition are in parts by mass: 0.01 - 0.1 part of DSHA, 0.05 - 0.2 part of pseudostellarin B, and 1.0 - 15.0 parts of alfalfa extract.

[0008] Preferably, the active ingredients of the anti-aging composition are in parts by mass: 0.03 - 0.08 part of DSHA, 0.08 - 0.15 part of pseudostellarin B, and 5.0 - 10.0 parts of alfalfa extract.

[0009] The alfalfa extract is prepared by pulsed ultrasonic-assisted subcritical extraction method, specifically: the dried alfalfa stems and leaves are crushed, added with water and subjected to pulsed ultrasonic extraction at room temperature, and then added with water and subjected to subcritical extraction at a temperature of 120 - 140 °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, added with glycerol 1 - 1.5 times the volume of the concentrated solution, stirred and mixed evenly, and then filtered to obtain the alfalfa extract. 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] Preparation method: Mix DSHA, pseudostellarin B and alfalfa extract, and dissolve them in purified water to make up 100 parts.

[0011] In the second aspect of the present invention, an application of the above anti-aging composition in the preparation of anti-aging skin care products is provided. Through experiments, it is verified that the above composition can promote the production of type I collagen and inhibit cell aging by increasing the protein expression levels of retinoic acid-binding receptors RARα and RXRγ, thereby achieving the anti-aging effect. It is also verified through experiments that the composition can significantly improve the anti-corrosion efficiency of the skin care product and reduce the dosage of preservatives in the skin care product.

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

[0013] The anti-aging skin care product may also contain other raw material components allowed to be added in any skin care product field, including but not limited to emulsifiers, emollients, moisturizers, coolants, skin conditioners, thickeners, preservatives, etc.

[0014] At the same time, by reasonably adding the above raw material components, different cosmetic dosage forms can also be prepared, such as essence water, essence milk, essence cream, etc., which are not specifically limited herein.

[0015] The beneficial technical effects of the present invention:

[0016] (1) The alfalfa extract of the present invention is obtained by pulsed ultrasound-assisted subcritical extraction technology. Compared with the traditional water extraction and alcohol precipitation method, it protects heat-sensitive components, prevents the decomposition of heat-sensitive components during the extraction process, and enables them to exert a better anti-aging effect; water is used as the extraction solvent without using organic solvents, which is more environmentally friendly.

[0017] (2) The anti-aging composition obtained by the above technical solution can significantly increase the content of retinoic acid-binding receptors RARα and RXRγ proteins and type I collagen, inhibit cell aging, and achieve an anti-aging effect similar to that of retinol. However, compared with retinol, it is mild and non-irritating;

[0018] (3) The anti-aging composition prepared by the above technical solution has good anti-corrosion performance in skin care products and can reduce or replace the use of traditional preservatives. Description of the Drawings

[0019] Figure 1 It is a gradient dilution diagram of bacteria;

[0020] Figure 2 It is a gradient dilution diagram of fungi. Detailed Embodiments

[0021] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0022] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0023] The present invention will be further described in conjunction with specific examples below. The following examples are only for explaining the present invention and do not limit its content. If the specific experimental conditions are not indicated in the examples, they are usually in accordance with conventional conditions or the conditions recommended by reagent companies; the reagents, consumables, etc. used in the following examples can be obtained from commercial sources without special instructions.

[0024] The present invention is further explained by the following examples, but it does not constitute a limitation of the present invention. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The test methods for which specific conditions are not specified in the following examples are usually carried out under conventional conditions. It should be noted that the DSHA in the examples and comparative examples of the present invention comes from Shandong Furuida Biological Co., Ltd., with a trade name of silanized hyaluronic acid, INCI name: dimethylsilanol hyaluronate; the Pseudostellariae Cyclotide B in the examples and comparative examples is purchased from Shanghai Xiaowei Biotechnology Co., Ltd., with a trade name of PortaVita TM HB Pseudostellariae Cyclotide; hyaluronic acid (3w-5w Da) in the comparative example is purchased from Shandong Focus Furuida Biological Co., Ltd.; retinol in the comparative example is purchased from Nanjing Anyike Chemical Co., Ltd.

[0025] In the embodiments and comparative examples of the present invention, the method for obtaining the alfalfa extract is:

[0026] 1. Water extraction and alcohol precipitation method: crush the dried alfalfa and pass it through a 60-mesh sieve, add water and reflux to extract twice, with a solid-liquid ratio of 1:10, and 2 hours each time. The combined extract is concentrated under reduced pressure to about 3 times the amount of medicinal materials. Add an appropriate amount of 95% ethanol solution to the concentrate until the alcohol content reaches 70% (w / w), stir for 10 minutes, and refrigerate (4±2℃) for more than 12 hours. Use a sterilizing filter plate and PP membrane for reduced pressure filtration, and the filtrate is decompressed and ethanol is recovered at 60℃, concentrated until there is no alcohol taste, and continued to concentrate to about 3 times the amount of medicinal materials, add glycerol 1.8 times the mass of the concentrate, stir and mix evenly, and filter to obtain alfalfa extract. In terms of crude drug amount, the concentration of alfalfa extract is 0.1227g / g.

[0027] 2. Pulse ultrasound assisted subcritical extraction method:

[0028] (1) The dried purple alfalfa stems and leaves were crushed and passed through a 60-mesh sieve, and water was added and extracted twice using pulse ultrasound (working 2s / resting 1s), each time with a solid-liquid ratio of 1:10, an ultrasonic power of 400W, a temperature of 25°C, and each time for 30 minutes. After the ultrasonication is completed, the extraction is set aside;

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

[0030] Examples 1 - 3:

[0031] The specific schemes of Examples 1 - 3 and Comparative Examples 1 - 11 are shown in Table 1 below, calculated by mass parts. The preparation method is: according to the formula in Table 1, mix DSHA, heterophyllin B, alfalfa extract, etc. according to the dosages in each example and comparative example, and dissolve them in purified water. Since retinol (VA) is insoluble in water, DMSO is used for dispersion and dissolution when conducting in vitro experimental evaluations.

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

[0033]

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

[0035] Prepare essence water by adding DSHA, heterophyllin B, and alfalfa extract according to the formula in Table 2 as Example 4. Comparative Example 12 is essence water using a classic preservative system, and Comparative Example 13 is essence water without the anti - aging composition compared to Example 4. Note: The raw materials in Table 2 are all sterile, and the preparations of Example 4 and Comparative Examples 12 and 13 are all completed under sterile conditions.

[0036] Table 2 List of components of specific examples and comparative examples for evaluating the preservative 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 °C and stir to dissolve. After complete dissolution, add phase B. After complete dissolution, start to cool down;

[0041] S2: Add Phase C when the temperature drops to 45 °C, stir to dissolve and disperse evenly to obtain the essence aquatic product.

[0042] Experimental Example 1: Irritation Test

[0043] Refer to SN / T 2329-2009 "Cosmetics - Eye Irritation / Chorioallantoic Membrane Test of Chicken Embryo" to conduct eye irritation test (i.e., safety test) on the product.

[0044] Experimental method: Use fertilized chicken embryos within 7 days old and incubate them in an incubator at 37.6 ± 0.5 °C and 50% - 70% humidity for 9 days.

[0045] Preparation of CAM: Conduct candling inspection, mark the position of the air chamber on the eggshell surface, use forceps to peel off the marked part of the eggshell to expose the white egg membrane. Be careful not to damage the integrity of the egg membrane during operation. Drop 0.5 mL of 0.9% NaCl solution to moisten the egg membrane thoroughly, gently blot the surface liquid with a tissue paper, and carefully remove the inner membrane with forceps to ensure that the vascular membrane is not damaged.

[0046] Reaction time method: Take 0.3 mL of the transparent test substance and evenly drip or smear it on the surface of the CAM. After acting for 3 minutes, wash off the test substance with 0.9% NaCl solution, observe the degree of change of each toxic effect of the CAM, and record the time when each toxic effect appears within 5 minutes. Set 6 chicken embryos for each test substance, and set 1 chicken embryo for the negative control (normal saline) and the positive control (1% SDS) respectively. The scoring criteria and phenomena are shown in Table 3.

[0047] Table 3 Scoring Criteria and Phenomena

[0048]

[0049]

[0050] Stimulation scoring method: Conduct the test using the reaction time method, and calculate the stimulation score (IS) using the following formula, with the result reserved to two decimal places:

[0051]

[0052] In the formula:

[0053] secH (bleeding time) ----- The average time when bleeding starts to occur observed on the CAM membrane, in seconds (s);

[0054] secL (vascular lysis time) ----- The average time when vascular lysis starts to occur observed on the CAM membrane, in seconds (s);

[0055] secC (clotting time) ----- The average time when clotting starts to appear observed on the CAM membrane, in seconds (s).

[0056] Classify the eye irritation of the test substance according to the IS value in Table 4.

[0057] Table 4 Evaluation of the results of the irritation scoring method

[0058] Stimulation score Stimulation classification IS<1 Non-irritating 1 ≤ IS < 5 Mild irritation 5 ≤ IS < 10 Moderate irritation IS ≥ 10 Strong irritation / corrosion

[0059] As can be seen from Table 5 of the test results, the chorioallantoic membrane tests of the anti-aging compositions in Examples 1 to 3 and Comparative Examples 1 to 6, and Comparative Examples 8 to 11 showed no irritation, indicating that one, two, or three combinations of DSHA, cyclopeptide B from Pseudostellaria heterophylla, and alfalfa extract are all safe, non-toxic, and have little irritation. The IS value of Comparative Example 7 was 8.42, which was moderately irritating (in Comparative Example 7, it was prepared by dissolving in olive oil, and the retinol content was 0.05%). This shows that the combined use of DSHA, cyclopeptide B from Pseudostellaria heterophylla, and alfalfa extract can reduce irritation compared with retinol.

[0060] Table 5 Statistical table of irritation test results

[0061]

[0062]

[0063] Test Example 2: In vitro experiment on promoting the activities of retinoic acid-binding receptors RAR-α and RXR-γ

[0064] In this experiment, human skin fibroblasts (HSF) (purchased from Guangdong Boxi Biotechnology Co., Ltd., batch number: 20081902, the same below) were treated with H2O2, and the expression levels of RAR-α and RXR-γ in the samples were detected by ELISA method 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 plan in Table 6, when the plating rate of fibroblasts in the 12-well plate reached 40% - 60%, HSF was treated with 400 μM H2O2 for 24 h. Grouped administration was carried out according to the test grouping, 2 mL of medicine was added to each well, and 3 replicate wells were set in each group. The blank group and the model group were added with 2 mL of 10% FBS complete medium (Lanzhou Rongye), and the sample group was added with 2 mL of complete medium containing the corresponding concentration of the test substance and 10% FBS. After the administration was completed, the 12-well plate was placed in an incubator (37 °C, 5% CO2) and cultured for 24 h. Detection was carried out according to the operation instructions of the ELISA kit (Abcam, the same below). The test results are shown in Tables 7 and 8.

[0065] Table 6 Test plan

[0066]

[0067] Note: The sample concentration is 2 mg / mL (diluted with deionized water. In Comparative Example 7, it was dissolved in DMSO to prepare a 0.05% retinol solution, and the sample loading concentration of the solution was 2 mg / mL).

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

[0069]

[0070] Note: ## indicates p < 0.01 compared with the blank group; ** indicates p < 0.01 compared with the model group; * indicates 0.01 < p < 0.05 compared with the model group.

[0071] It can be seen from the test results in Table 7 that Comparative Examples 3 and 10 show that the single alfalfa extract does not have the effect of promoting RAR-α expression. Comparative Examples 1 and 8 show that the use of single DSHA and sodium hyaluronate (3w - 5w Da) has a certain promoting effect on the RAR-α receptor; Comparative Example 9 shows that the synergistic effect of sodium hyaluronate (3w - 5w Da), cyclopeptide B from Pseudostellaria heterophylla and alfalfa extract 1 is not obvious; Comparative Example 11 shows that the synergistic effect of DSHA, cyclopeptide B from Pseudostellaria heterophylla and alfalfa extract 2 is not as obvious as that of the Examples. Examples 1 - 3 can all significantly up-regulate the content of RAR-α, indicating that DSHA, cyclopeptide B from Pseudostellaria heterophylla and alfalfa extract 1 have a significant synergistic promoting effect. At the same time, the efficacy of Example 2 is equivalent to that of Comparative Example 7 (single retinol).

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

[0073]

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

[0075] It can be seen from Table 8 that Comparative Examples 1, 3, 8, and 10 show that single DSHA, sodium hyaluronate (3w - 5w Da), and alfalfa extract have a certain promoting effect on the expression of the RXR-γ receptor; Examples 1 - 3 can all significantly up-regulate the gene expression of RXR-γ. At the same time, the efficacy of Example 2 is equivalent to that of Comparative Example 7 (single retinol), indicating that DSHA, cyclopeptide B from Pseudostellaria heterophylla and alfalfa extract 1 have a significant synergistic promoting effect. The effect of Comparative Example 9 is lower than that of Examples 1 - 3, indicating that sodium hyaluronate (3w - 5w Da), cyclopeptide B from Pseudostellaria heterophylla and alfalfa extract 1 do not have a significant synergistic promoting effect. When Example 2 is compared with Comparative Example 11, the synergistic effect of alfalfa extract 1 with DAHA and cyclopeptide B from Pseudostellaria heterophylla is better than the synergistic effect of alfalfa extract 2.

[0076] Experimental Example 3: Experiment on the proportion of senescent cells after in vitro testing of β-galactosidase staining

[0077] The β-galactosidase staining method was used to detect the effect of the anti-aging composition on the level of cell senescence; the β-galactosidase staining method included the following steps: According to the test grouping in Table 9, when the confluence rate of HSF in the 6-well plate reached 40% - 60%, it was stimulated with 300 μM H2O2 for 2 h. Grouped administration was carried out according to the test grouping, 2 mL of sample was added to each well, and 3 replicates were set for each group. 2 mL of DMEM culture medium containing 5% serum (Thermo Fisher, the same below) was added to each well of the blank control group and the negative control group, 2 mL of DMEM culture medium containing 50 ng / ml TGF-β1 and 5% serum was added to each well of the positive control group, and 2 mL of DMEM culture medium containing the corresponding concentration of the test substance and 5% serum was added to each well of the sample group. After the administration was completed, the 6-well plate was placed in an incubator and cultured for 22 h. The cells were digested with 0.25% trypsin (Thermo Fisher), inoculated into a 6-well plate, incubated in an incubator for 24 h, the cell culture medium was aspirated, washed once with PBS, 1 mL of β-galactosidase staining fixative was added, and fixed at room temperature for 15 min. The cell fixative was aspirated, and the cells were washed 3 times with PBS. The PBS was aspirated, 1 mL of staining working solution was added to each well, incubated overnight at 37 °C, the 6-well plate was sealed with a sealing film to prevent evaporation, observed and photographed under an optical microscope, and the inhibition rate results are shown in Table 10. The inhibition rate calculation formula is as follows:

[0078]

[0079] Table 9 Test scheme

[0080]

[0081]

[0082] Note: The sample concentration was 2 mg / mL (diluted with deionized water, and in Comparative Example 7, retinol was dissolved in DMSO to prepare a 0.05% solution, and the solution loading concentration was 2 mg / mL).

[0083] Table 10 Test results of fibroblast senescence level

[0084]

[0085] Note: ## indicates p < 0.01 compared with the blank group; ** indicates p < 0.01 compared with the blank group; * indicates 0.01 < p < 0.05 compared with the blank group.

[0086] As can be seen from the results in Table 10, the results of Comparative Examples 1-11 show that the use of DSHA, sodium hyaluronate (3w-5w Da), cycloastragenol B, and Medicago sativa extract alone or in combination has a certain inhibitory effect on fibroblast senescence, while the use of retinol alone has no obvious effect on improving cell senescence; Examples 1-3 can significantly reduce the positive cell rate after β-galactosidase staining, and it is positively correlated with the concentration, indicating that the combination of DSHA, cycloastragenol B, and Medicago sativa extract 1 can significantly inhibit fibroblast senescence. The inhibitory ability of Comparative Examples 9 and 11 is lower than that of Examples 1-3, indicating that the synergistic effect among sodium hyaluronate (3w-5w Da), cycloastragenol B, and Medicago sativa extract is not obvious, and the synergistic effect of Medicago sativa extract 1 is better than that of Medicago sativa extract 2.

[0087] Test Example 4: Experiment on Promoting the Synthesis of Type I Collagen

[0088] In this experiment, HSF was irradiated with UVA to detect the change in the content of type I collagen (CollagenⅠ) in the samples. According to the test protocol in Table 11, when the plating rate of fibroblasts in the 24-well plate reached 40%-60%, grouping and drug administration were carried out, with 2 mL of sample added to each well, and 3 replicate wells were set in each group. The blank control group and the negative control group added 2 mL of DMEM medium containing 5% serum to each well, the positive control group added 2 mL of DMEM medium containing 100 ng / ml TGF-β1 and 5% serum to each well, and the sample group added 2 mL of DMEM medium containing the corresponding concentration of the test substance and 5% serum to each well. After drug administration, the 6-well plate was placed in an incubator (37°C, 5% CO2) and cultured for 24 h. According to the test grouping, the groups that needed UVA irradiation were irradiated with UVA at 30 J / cm 2 for 26 min and then continued to be cultured in an incubator (37°C, 5% CO2) for 24 h. Detection was carried out according to the operation instructions of the ELISA kit. The test results are shown in Table 12.

[0089] Table 11 Test Protocol

[0090]

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

[0092] Table 12 Summary Table of CollagenⅠ Content Results

[0093]

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

[0095] It can be seen from Table 12 that Comparative Examples 1, 2, and 8 show that the use of DSHA alone, sodium hyaluronate (3w - 5w Da), and cyclopeptide B of Pseudostellaria heterophylla has a certain promoting effect on the production of type I collagen. The promoting effect of using a single alfalfa extract in Comparative Examples 3 and 11 is not obvious. Examples 1 to 3 can all significantly promote the production of type I collagen, and the promoting effect of Example 2 is the most significant, indicating that the combination of DSHA, cyclopeptide B of Pseudostellaria heterophylla, and alfalfa extract 1 can significantly promote the production of type I collagen and has a synergistic effect.

[0096] Test Example 5: Antiseptic Efficacy Evaluation Experiment

[0097] Experimental Strains:

[0098] 1. Bacteria: Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, all of which are the 3rd generation cultures;

[0099] 2. Fungi: Aspergillus niger, Candida albicans, all of which are the 3rd generation cultures.

[0100] Experimental Procedures:

[0101] 1. Preparation of Bacterial Suspension: The cryopreserved strains of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa mentioned above were respectively inoculated into tryptic soy broth liquid medium and cultured at 32°C for 48 hours. They were serially diluted with 0.85% normal saline. 2 ml of the diluted solution was taken from each dilution gradient and injected into two sterilized petri dishes, 1 ml per petri dish. Tryptic soy agar medium was poured onto the plates and cultured at 32°C for 48 hours for counting. Bacterial suspensions with a 10 8 order of magnitude were mixed in equal volumes for standby.

[0102] 2. Preparation of Candida albicans Suspension:

[0103] The cryopreserved strain of Candida albicans mentioned above was inoculated into Sabouraud dextrose liquid medium and cultured at 28°C for 5 days. It was serially diluted with 0.85% normal saline. 2 ml of the diluted solution was taken from each dilution gradient and injected into two sterilized petri dishes, 1 ml per petri dish. Sabouraud dextrose agar medium was poured onto the plates and cultured at 28°C for 5 days for counting. A bacterial suspension with a 10 6 order of magnitude was used for standby.

[0104] 3. Preparation of Aspergillus niger Spore Suspension:

[0105] Inoculate the cryopreserved spores suspension of Aspergillus niger mentioned above onto Sabouraud dextrose agar slants, and culture at 28°C for 5 - 7 days. Take 25 ml of 0.85% normal saline containing 0.05% Tween 80 to rinse the bacterial lawn to prepare a spores suspension. Perform gradient dilution with 0.85% normal saline containing 0.05% Tween 80. Pipette 2 ml of the dilution from each gradient and inject them into two sterilized Petri dishes, 1 ml per dish. Pour plates with Sabouraud dextrose agar medium and culture at 28°C for 72 hours, then count. Select a spore suspension of the 10 6 order of magnitude for standby.

[0106] 4. Gradient dilution diagram

[0107] 1) The gradient dilution diagram of bacteria is as shown in Figure 1 the figure.

[0108] 2) The gradient dilution diagram of fungi is as shown in Figure 2 the figure.

[0109] 5. Sample preparation: Weigh 3 portions of each sample, 120 g for each portion, and place them in sterilized conical flasks for standby.

[0110] 6. Sample inoculation: Take the 3 weighed samples mentioned above and inoculate them with 1.2 ml of the standby bacterial mixed suspension, 1.2 ml of Candida albicans suspension, and 1.2 ml of Aspergillus niger spore suspension respectively. Make marks, mix well, and store the samples at room temperature.

[0111] 7. Sample detection: Take samples from the above - prepared samples on the 0th, 1st, 3rd, 7th, 14th, 21st, and 28th days respectively, and perform detection according to the cosmetic microbiological inspection procedure. Record the number of colonies in the samples to judge the anti - corrosion efficacy of the cosmetics.

[0112] 8. Results of bacterial suspension counting:

[0113] The counting results of the bacterial suspension are shown in Table 13. The initial inoculation concentration of the sample is: 1.4×10 6 CFU / g (initial logarithm value 6.15).

[0114] Table 13 Counting results of bacterial suspension

[0115]

[0116]

[0117] The counting results of the Candida albicans suspension are shown in Table 14. The initial inoculation concentration of the sample is: 1.5×10 4 CFU / g (initial logarithm value 4.18).

[0118] Table 14 Counting results of Candida albicans suspension

[0119] Name of strain Counting result Candida albicans <![CDATA[1.5×10 6 CFU / ml]]>

[0120] The counting results of the Aspergillus niger spore suspension are shown in Table 15. The initial inoculation concentration of the sample was: 1.2×10 4 CFU / g (initial logarithm value 4.08).

[0121] Table 15 Counting results of Aspergillus niger spore suspension

[0122] Name of strain Counting result Aspergillus niger <![CDATA[1.2×10 6 CFU / ml]]>

[0123] The anti-corrosion effect of cosmetics is evaluated by the logarithmic reduction value. The calculation method of this index is shown in Equation 1.

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

[0125] Wherein:

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

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

[0128] The evaluation criteria for the anti-corrosion effect of cosmetics are shown in Table 16, and the counting results of the samples are shown in Table 17.

[0129] Table 16 Evaluation criteria for the anti-corrosion effect of cosmetics

[0130]

[0131] Table 17 Counting results of samples

[0132]

[0133]

[0134] Comparative Example 12 is a classic anti-corrosion system. According to the results in Table 17, it can be seen that both Example 4 and Comparative Example 12 passed the anti-corrosion efficacy standard test, and the anti-corrosion efficacy of Example 4 against bacteria and Aspergillus niger is better than that of Comparative Example 12; Comparative Example 13 did not pass the anti-corrosion efficacy evaluation experiment. It shows that the combined use of DSHA, heterophyllin and Medicago sativa extract 1 can achieve a certain synergistic effect in promoting anti-corrosion efficacy, reduce the dosage of preservatives in the formula, and thus reduce the irritation of preservatives to the skin.

[0135] It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the given examples, those of ordinary skill in the art can modify or equivalently replace the technical solution of the present invention as needed, without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. An anti-aging composition, characterized in that, The active ingredients of the anti-aging composition are in parts by mass: 0.01 - 0.1 part of DSHA, 0.05 - 0.2 part of cyclopeptide B from Pseudostellaria heterophylla, and 1.0 - 15.0 parts of Medicago sativa extract; the DSHA is dimethylsilanol hyaluronate.

2. The anti-aging composition according to claim 1, wherein The active ingredients of the anti-aging composition are in parts by mass: 0.03 - 0.08 part of DSHA, 0.08 - 0.15 part of cyclopeptide B from Pseudostellaria heterophylla, and 5.0 - 10.0 parts of Medicago sativa extract.

3. The anti-aging composition according to claim 1 or 2, characterized in that, The Medicago sativa extract is obtained by pulsed ultrasonic-assisted subcritical extraction method, 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.

4. The anti-aging composition according to claim 3, wherein The preparation method of the Medicago sativa extract is as follows: crush the dried stems and leaves of Medicago sativa, add water and carry out pulsed ultrasonic extraction at room temperature, then continue to add water and carry out subcritical extraction at a temperature of 120 - 140 °C and a pressure of 0.8 - 1.2 MPa, concentrate the filtrate under reduced pressure to 4 - 6 times the mass of the Medicago sativa powder, add glycerol 1 - 1.5 times the volume of the concentrated solution, stir and mix evenly, and obtain the Medicago sativa extract after filtration.

5. The anti-aging composition according to claim 1 or 2, characterized in that, Mix DSHA, cyclopeptide B from Pseudostellaria heterophylla and Medicago sativa extract, and dissolve them in purified water to make up 100 parts.

6. Use of the anti-aging composition according to claim 1 or 2 in the preparation of anti-aging skin care products.

7. The application according to claim 6, characterized in that, By increasing the protein expression levels of retinoic acid-binding receptors RARα and RXRγ, promoting the production of type I collagen, and inhibiting cell aging, thereby achieving an anti-aging effect, and the use of the anti-aging composition reduces the amount of preservatives used.

8. An anti-aging skin care product, characterized in that, Using the anti-aging composition according to claim 1 or 2 as the active ingredient.

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