Composition with skin anti-aging effect and application thereof

Abstract

The invention belongs to the technical field of cosmetics, and particularly relates to a composition with a skin anti-aging effect and application thereof. The composition comprises micromolecular collagen peptide and calendula exosome, and the weight ratio of the micromolecular collagen peptide to the calendula exosome is (1-10): (10-1). In the composition disclosed by the invention, the micromolecular collagen peptide and the calendula exosome in a specific dosage ratio are combined, so that the composition disclosed by the invention has synergistic effects of moisturizing, repairing a skin barrier and resisting wrinkles and tightening, and when the composition is applied to cosmetics, the skin cuticle and barrier function reconstruction can be promoted; the moisturizing property and the water resistance of the skin are improved, the wrinkles of the skin are reduced, the skin is more compact, and the use feeling is excellent.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and in particular relates to a composition with skin anti-aging effect and application thereof. Background Art

[0002] Aging, long-term exposure to adverse environmental factors, malnutrition, fatigue, and other factors can alter the skin's visual appearance, physical properties, or physiological functions. The most significant and noticeable changes include the formation of fine lines and wrinkles, decreased elasticity, sagging skin, decreased firmness, uneven or dull skin tone, rough surface texture, and the appearance of pigmentation. Changes that occur as the skin ages or experiences long-term environmental damage include a general decrease in cell and tissue activity, a decrease in cell replication rate, decreased blood flow to the skin, decreased moisture content, an accumulation of structural and functional errors, changes in skin metabolism, and a decrease in the skin's ability to rebuild and repair itself. Many changes in the skin's appearance and barrier function are primarily caused by changes in the skin's outer epidermal layer, while other changes, such as wrinkles and sagging, are primarily caused by changes in the underlying dermis.

[0003] In order to prevent these phenomena and maintain healthier and more elastic skin, the present inventors have been committed to researching and developing cosmetics containing various highly active substances to maintain the inherent functions of the skin and activate skin cells, thereby effectively slowing down skin aging.

[0004] Small molecule collagen peptides are low molecular weight bioactive peptides with special functions obtained through deep hydrolysis. Compared with proteins, small molecule collagen peptides have a higher human absorption rate and stronger biological activity. At the same time, they are highly safe and have good biocompatibility. Based on these excellent properties, people are increasingly favoring the use of small molecule collagen peptides to delay skin aging. However, in actual applications, it is found that even though the molecular weight of small molecule collagen peptides is small, they still need to penetrate the stratum corneum and epidermis of the skin to reach the dermis and exert their effects. The barrier function of the skin itself will hinder the penetration of active substances. Therefore, most of the currently commercialized collagen peptides have low actual absorption rates and unsatisfactory results.

[0005] In recent years, plant-derived vesicle-like particles (PDEVLP) have garnered increasing attention due to their potential bioactive properties, becoming a research hotspot in the fields of biomedicine and skincare. Numerous studies have identified and reported the presence of PDEVLP in vegetables, fruits, and medicinal plants. PDEVLP's wide availability, high yield, simple production, and minimal immunogenicity risks make it a safer and more economical option for development as a variety of functional active substances or carriers.

[0006] In response to the above-mentioned defects in the prior art, the inventors have developed a composition with skin anti-aging effects that has moisturizing and skin barrier repair as well as anti-wrinkle and firming effects. It can be conveniently used in various cosmetics to give the target cosmetics the above-mentioned effects. Summary of the Invention

[0007] The present invention aims to overcome the shortcomings of the prior art by providing a skin-anti-aging composition with moisturizing, skin barrier repair, and anti-wrinkle and firming properties, as well as a preparation method and use thereof. The present composition effectively addresses the low absorption rate and unsatisfactory effects of small-molecule collagen peptides by combining them with marigold exosomes in a specific dosage ratio. This results in synergistic moisturizing, skin barrier repair, and anti-wrinkle and firming properties.

[0008] Specifically, the present invention is achieved through the following technical solutions:

[0009] In a first aspect, the present invention provides a composition with anti-aging effects on the skin, comprising small molecule collagen peptides and marigold exosomes, wherein the weight ratio of the small molecule collagen peptides to the marigold exosomes is 1-10:10-1.

[0010] As an optional manner, in the above composition, the weight ratio of the small molecule collagen peptide to the marigold exosomes is 1-8:8-1.

[0011] As an optional manner, in the above composition, the weight ratio of the small molecule collagen peptide to the marigold exosomes is 1-4:4-1.

[0012] As an optional manner, in the above composition, the weight ratio of the small molecule collagen peptide to the marigold exosomes is 2:3.

[0013] As an optional mode, in the above composition, the composition consists of small molecule collagen peptides and marigold exosomes.

[0014] As an optional manner, in the above composition, the small molecule collagen peptide is separated and identified from the enzymatic hydrolysate of deep-sea cod skin, and the amino acid sequence of the small molecule collagen peptide is HPTAE (His-Pro-Thr-Ala-Glu).

[0015] As an optional manner, in the above composition, the small molecule collagen peptide is prepared by solid phase synthesis.

[0016] As an optional manner, in the above composition, the marigold exosomes have a double-membrane structure under a transmission electron microscope, mostly in the shape of a concave round cake, and the particle size of the marigold exosomes is 50-300 nm.

[0017] As an optional method, in the above-mentioned composition, the primary product of marigold exosomes can be prepared by differential centrifugation and sedimentation, or by ultrafiltration, or by kit extraction, and then purified by at least one of density gradient, column chromatography, ultrafiltration, etc.

[0018] As an optional method, in the above composition, the preparation method of the marigold exosomes comprises the following steps:

[0019] (1) Fresh marigolds washed with pure water were soaked in an appropriate amount of PBS, and after being fully soaked, they were crushed in a wall-breaking machine, and filtered through a gauze to obtain filtrate a;

[0020] (2) centrifuging the filtrate a at 1,000 g for 10-15 min to obtain supernatant liquid a, and centrifuging the supernatant liquid a at 3,000 g for 30-40 min to obtain supernatant liquid b;

[0021] (3) Supernatant b was concentrated by centrifugation at 3,500 g for 30-40 min using a 100 kDa ultrafiltration tube to obtain supernatant concentrate c, and supernatant concentrate c was centrifuged at 10,000 g for 30-40 min to obtain supernatant concentrate d;

[0022] (4) Filter the supernatant concentrate d through a 0.45 μm filter membrane to obtain filtrate b;

[0023] (5) The filtrate b is subjected to ultrahigh-speed centrifugation at 100,000 to 120,000 g for 60 to 90 minutes to obtain a precipitate, thereby obtaining the marigold exosomes.

[0024] As an optional method, in the above composition, the method for preparing the marigold exosomes comprises the following steps:

[0025] (1) Fresh marigolds washed with pure water were soaked in an appropriate amount of PBS, and after being fully soaked, they were crushed in a wall-breaking machine, and filtered through a gauze to obtain filtrate a;

[0026] (2) Filtrate a was centrifuged at 1,000 g for 12 min to obtain supernatant liquid a, and supernatant liquid a was centrifuged at 3,000 g for 30 min to obtain supernatant liquid b;

[0027] (3) Supernatant b was concentrated by centrifugation at 3,500 g for 30 min using a 100 kDa ultrafiltration tube to obtain supernatant concentrate c, and supernatant concentrate c was centrifuged at 10,000 g for 30 min to obtain supernatant concentrate d;

[0028] (4) Filter the supernatant concentrate d through a 0.45 μm filter membrane to obtain filtrate b;

[0029] (5) The filtrate b was subjected to ultrahigh-speed centrifugation at 120,000 g for 70 min to obtain a precipitate, thereby obtaining the marigold exosomes.

[0030] As an optional method, in the above composition, the small molecule collagen peptide and the marigold exosomes are resuspended in a sterilized filtered PBS solution, mixed evenly, and then freeze-dried.

[0031] In a second aspect, the present invention provides use of the composition with skin anti-aging effects described in the first aspect in the preparation of cosmetics with moisturizing and skin barrier repairing, as well as anti-wrinkle and firming effects.

[0032] As an optional mode, in the above use, the cosmetic is toner, lotion, essence, cream, ointment, facial mask or freeze-dried powder.

[0033] As an optional mode, in the above-mentioned use, the cosmetic has a protective effect on UV-induced cell damage, has antioxidant activity, repairs damaged cells, protects the skin barrier, and delays skin aging.

[0034] As an optional mode, in the above use, the content of the composition in the cosmetic is 1%-5% by mass.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] (1) The inventors screened the enzymatic hydrolysates of deep-sea cod skin and obtained for the first time a small molecule collagen peptide HPTAE (His-Pro-Thr-Ala-Glu) that has the functions of protecting cell activity, inhibiting oxidative damage, protecting the skin barrier, enhancing collagen synthesis, inhibiting collagen degradation, repairing skin damage and having no obvious side effects.

[0037] (2) In the composition of the present invention, by combining small molecule collagen peptides and marigold exosomes in a specific dosage ratio, the problem of low absorption rate of small molecule collagen peptides and unsatisfactory use effect is well solved, so that the composition of the present invention has synergistic moisturizing and skin barrier repair as well as anti-wrinkle and firming effects.

[0038] (3) The composition of the present invention is applied to cosmetics, which can promote the reconstruction of the skin's stratum corneum and barrier function, improve the skin's moisturizing and water resistance, reduce skin wrinkles and make it tighter, and provide an excellent user experience. DETAILED DESCRIPTION

[0039] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0040] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0041] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0042] Unless otherwise specified, all percentages and parts in the present invention are by weight.

[0043] Preparation Example:

[0044] 1. Preparation of small molecule collagen peptide HPTAE (His-Pro-Thr-Ala-Glu) 1.1 Preparation of small molecule collagen peptide

[0045] An appropriate amount of frozen deep-sea cod skin was taken, and water was added to thaw it in a ratio of 1:3 (w / v). Then, 5% NaOH was added to soak it to remove impurities, and it was rinsed with clean water several times until the pH of the washing water was neutral. 0.06% alkaline protease and 0.04% bromelain were added, and enzymatic hydrolysis was carried out for 5 hours. Then, the enzyme was inactivated in a boiling water bath for 15 minutes. The enzymatic hydrolysis solution was passed through a 200-mesh silk cloth to remove impurities, and suspended impurities were removed by filtration. The solution was then centrifuged at 7500 rpm and 4°C for 10 minutes. The supernatant was taken and freeze-dried to obtain the deep-sea cod skin enzymatic hydrolysis product.

[0046] 1.2 Screening and identification of small molecule collagen peptide sequences

[0047] The deep-sea cod skin enzymatic hydrolysate prepared in step 1.1 above was desalted and then subjected to chromatographic separation and mass spectrometry detection.

[0048] Specific conditions are as follows: LC-MS / MS analysis was performed using a dual ternary high-performance liquid chromatography coupled to a quadrupole-orbitrap high-resolution mass spectrometer. Reversed-phase separation was used, with 0.1% formic acid in water as the eluent (phase A) and acetonitrile as the eluent (phase B). The chromatographic column was a C18, 100 × 2.1 mm, Waters, with a flow rate of 0.2 mL / min, a column temperature of 40°C, and an injection volume of 2 μL. The mass spectrometer scan mode was positive ionization, with a primary mass spectrometer scan range of 150–1500 m / z, a resolution of 35,000, and a maximum injection time of 100 ms. The MS2 scan mode was 17,500 with a maximum injection time of 50 ms. The collision energy was 35 eV, the capillary spray voltage was 3.0 kV, the capillary temperature was 320°C, the auxiliary heater temperature was 350°C, and the S-lens Rf was set to 60. Ten fragmentation spectra (MS2 scans) were acquired after each full scan.

[0049] Database search: The mass spectrometry test raw file (Raw File) is searched into the corresponding database using the software MaxQuant, and finally the protein identification and quantitative analysis results are obtained.

[0050] Mass spectrometry was used to identify the peptide sequences of the enzymatic hydrolysates of deep-sea cod skin, and peptide sequences with relatively high relative content and high solubility, non-toxicity and good stability predicted by database search were selected for further research.

[0051] 1.3 Preparation of HPTAE (His-Pro-Thr-Ala-Glu) by solid-phase synthesis

[0052] The resin is swollen, washed, and drained, and the Fmoc protecting group is removed. The amino acids that make up the peptide are weighed and condensed in a shaker at 30°C for 1 hour. The removal-protection-condensation process is repeated until all amino acids are connected. The target peptide is separated from impurities using high-performance liquid chromatography (HPLC) to obtain a pure peptide (>99%). The target peptide is then lyophilized into a powder.

[0053] In addition, the same method was used to synthesize the small molecule collagen peptide HPSAE (His-Pro-Ser-Ala-Glu) used as a control in the following "Effect Examples" section.

[0054] 2. Preparation of Calendula Exosomes

[0055] The preparation method of marigold exosomes comprises the following steps:

[0056] (1) Fresh marigolds washed with pure water were soaked in an appropriate amount of PBS, and after being fully soaked, they were crushed in a wall-breaking machine, and filtered through a gauze to obtain filtrate a;

[0057] (2) Filtrate a was centrifuged at 1,000 g for 12 min to obtain supernatant liquid a, and supernatant liquid a was centrifuged at 3,000 g for 30 min to obtain supernatant liquid b;

[0058] (3) Supernatant b was concentrated by centrifugation at 3,500 g for 30 min using a 100 kDa ultrafiltration tube to obtain supernatant concentrate c, and supernatant concentrate c was centrifuged at 10,000 g for 30 min to obtain supernatant concentrate d;

[0059] (4) Filter the supernatant concentrate d through a 0.45 μm filter membrane to obtain filtrate b;

[0060] (5) The filtrate b was subjected to ultrahigh-speed centrifugation at 120,000 g for 70 min to obtain a precipitate, thereby obtaining the marigold exosomes.

[0061] In addition, the same method was used to prepare Rhodiola rosea exosomes as a control in the following “Effect Examples” section.

[0062] 3. Preparation of Example 1

[0063] A composition with skin anti-aging effects, comprising small molecule collagen peptide HPTAE (His-Pro-Thr-Ala-Glu) and marigold exosomes, wherein the weight ratio of the small molecule collagen peptide to the marigold exosomes is 2:3.

[0064] The small molecule collagen peptides and calendula exosomes were resuspended in a sterilized filtered PBS solution, mixed evenly, and then freeze-dried.

[0065] 4. Preparation of Example 2

[0066] A composition with skin anti-aging effects, comprising small molecule collagen peptide HPTAE (His-Pro-Thr-Ala-Glu) and marigold exosomes, wherein the weight ratio of the small molecule collagen peptide to the marigold exosomes is 1:4.

[0067] The small molecule collagen peptides and calendula exosomes were resuspended in a sterilized PBS solution, mixed evenly, and then freeze-dried.

[0068] 5. Preparation of Example 3

[0069] A composition with skin anti-aging effects, comprising small molecule collagen peptide HPTAE (His-Pro-Thr-Ala-Glu) and marigold exosomes, wherein the weight ratio of the small molecule collagen peptide to the marigold exosomes is 4:1.

[0070] The small molecule collagen peptides and calendula exosomes were resuspended in a sterilized filtered PBS solution, mixed evenly, and then freeze-dried.

[0071] 6. Preparation of Comparative Example 1

[0072] Only 2 parts by weight of the small molecule collagen peptide HPTAE (His-Pro-Thr-Ala-Glu) was used, resuspended in a filter-sterilized PBS solution, and lyophilized.

[0073] 7. Preparation of Comparative Example 2

[0074] Only 3 parts by weight of Calendula officinalis exosomes were used, resuspended in filter-sterilized PBS solution, and lyophilized.

[0075] 8. Preparation of Comparative Example 3

[0076] Only 2 parts by weight of the small molecule collagen peptide HPSAE (His-Pro-Ser-Ala-Glu) was used, resuspended in a filter-sterilized PBS solution, and lyophilized.

[0077] 9. Preparation of Comparative Example 4

[0078] Only 3 parts by weight of Rhodiola rosea exosomes were used, resuspended in filter-sterilized PBS solution, and lyophilized.

[0079] 10. Preparation of Comparative Example 5

[0080] A composition is composed of small molecule collagen peptide HPTAE (His-Pro-Thr-Ala-Glu) and Rhodiola rosea exosomes, wherein the weight ratio of the small molecule collagen peptide to the Rhodiola rosea exosomes is 2:3.

[0081] The small molecule collagen peptide and Rhodiola rosea exosomes were resuspended in a sterilized PBS solution, mixed evenly, and then freeze-dried.

[0082] 11. Preparation of Comparative Example 6

[0083] A composition is composed of small molecule collagen peptide HPSAE (His-Pro-Ser-Ala-Glu) and marigold exosomes, wherein the weight ratio of the small molecule collagen peptide to the marigold exosomes is 2:3.

[0084] The small molecule collagen peptides and calendula exosomes were resuspended in a sterilized filtered PBS solution, mixed evenly, and then freeze-dried.

[0085] 12. Preparation of Comparative Example 7

[0086] A composition is composed of small molecule collagen peptide HPTAE (His-Pro-Thr-Ala-Glu) and Rhodiola rosea exosomes, wherein the weight ratio of the small molecule collagen peptide to the Rhodiola rosea exosomes is 2:3.

[0087] The small molecule collagen peptide and Rhodiola rosea exosomes were resuspended in a sterilized PBS solution, mixed evenly, and then freeze-dried.

[0088] Effect embodiment:

[0089] Effect Example 1: Effect of the composition with skin anti-aging effect of the present invention on the survival rate of UV-damaged HACAT cells

[0090] The amount of endogenous adenosine triphosphate (ATP) in cells can reflect the activity of cells. ATP was used to quantitatively detect cell activity. The HACAT cells used in Example 1 were purchased from Wuhan Punosai Life Science Technology Co., Ltd. HACAT cells were cultured at a concentration of 1×10 4 The cells were seeded at a density of 1 / well in a 96-well plate and DMEM culture medium was added. Six replicates were set up for each group, and the blank group was not inoculated with cells, and the same volume of culture medium was added. After 24 hours of cultivation, the model control group and the sample group were UV modeled for 70 minutes, and the old culture medium was subsequently discarded. The blank control empty and model wells were replaced with fresh culture medium, and the sample groups (Example 1-3 groups and Comparative Example 1-7 groups) were replaced with culture medium containing the sample. After 18 hours of continuous cultivation, the old culture medium was discarded, 100 μL of ATP was added to each well, and the cells were treated at 37°C for 1 hour for lysis. The supernatant was taken to an enzyme-labeled plate, and the RLU value was determined using a chemiluminescence instrument. The results were analyzed using SPSS27.0 statistical software, and the data were expressed as mean ± standard deviation. One-way analysis of variance was used for comparison between multiple groups, and t-test was used for comparison between two groups. p<0.05 indicated that the difference was statistically significant.

[0091] The experimental results are shown in Table 1 below. Compared with the blank group, the RLU value of the model group was significantly increased, indicating the success of the modeling. The groups of Examples 1 to 3 all had a significant effect on increasing the RLU value of HACAT cells caused by UV damage ( *** p<0.001), among which the effect of Example 1 group was the most significant.

[0092] From the experimental results of the comparative group, it can be seen that the use of small molecule collagen peptide HPTAE (His-Pro-Thr-Ala-Glu) or Rhodiola rosea exosomes alone, or the combination of small molecule collagen peptide HPTAE (His-Pro-Thr-Ala-Glu) and Rhodiola rosea exosomes, also significantly increased the reduction in RLU value of HACAT cells caused by UV damage, but the effect was not as good as that of Examples 1 to 3. However, after the small molecule collagen peptide was used in combination with Rhodiola rosea exosomes, an adverse interaction was substantially generated between the two, and the effect was not as good as the use of the small molecule collagen peptide alone.

[0093] The comparative example of small molecule collagen peptide HPSAE (His-Pro-Ser-Ala-Glu) alone cannot reduce UV-induced HACAT cell damage, which indicates that the activity of small molecule collagen peptides is related to their specific amino acid sequence structure. In addition, marigold exosomes alone cannot reduce UV-induced HACAT cell damage, but when combined with the small molecule collagen peptide HPTAE (His-Pro-Thr-Ala-Glu) of the present invention, the survival rate of HACAT cells can reach the same level as that of the blank group, and its effect is better than the effect of the small molecule collagen peptide of the present invention combined with Rhodiola rosea exosomes, which have the ability to improve UV damage. This shows that the combination of specific small molecule collagen peptides and specific types of plant-derived exosomes has an unexpected and significant synergistic effect in reducing UV-induced HACAT cell damage.

[0094] Table 1: Effect of the composition with skin anti-aging effect of the present invention on the survival rate of UV-damaged HACAT cells (mean ± standard deviation)

[0095] Group RLU value Blank group <![CDATA[1590573±48756 *** ]]> Model Group 1418043±52371 Example 1 group <![CDATA[1600445±46182 *** ]]> Example 2 group <![CDATA[1563188±48225 *** ]]> Example 3 group <![CDATA[1582473±49358 *** ]]> Comparative Example 1 <![CDATA[1524986±51329 ** ]]> Comparative Example 2 1443596±47622 Comparative Example 3 1435762±46185 Comparative Example 4 <![CDATA[1471925±52615 * ]]> Comparative Example 5 <![CDATA[1510126±47153 ** ]]> Comparative Example 6 1441642±48326 Comparative Example 7 1451745±50314

[0096] Note: Compared with the model group, * p<0.05; ** p<0.01; *** p<0.001.

[0097] Effect Example 2: Test of the effect of the composition with skin anti-aging effect of the present invention on skin moisturizing

[0098] The skin moisture retention of 55 volunteers (5 per group) was tested using a Corneometer CM 825 (Courage Khazaka, Germany). A 4 cm × 4 cm test area was marked on the flexor side of the left and right forearms of the subjects. Each sample (containing, by mass, 2% of Examples 1 to 3 and Comparative Examples 1 to 7 and 98% of a conventional cosmetic base ingredient used in preparing facial creams) was applied at a rate of 3.0 mg / cm.2 , and gently massage until the sample is absorbed. Use a skin moisture meter to measure the skin moisture content before and 4 hours after application. Calculate the skin moisture increase rate according to the following formula. The average skin moisture increase rate of volunteers using each sample is the average skin moisture increase rate of that sample.

[0099] Skin moisture content increase rate (%) = (measured value after application - base value before application) / base value before application x 100%

[0100] The experimental results are shown in Table 2 below. As can be seen from the data in Table 2, the skin moisturizing effect of conventional cosmetic base ingredients used to prepare face creams is poor. After 4 hours, the average skin moisture content of volunteers in the face cream base group increased by only 6.25%. The composition with skin anti-aging effects of the present invention and the small molecule collagen peptide HPTAE (His-Pro-Thr-Ala-Glu) can both significantly increase the average skin moisture content of volunteers when used alone, and the effect is better when used in the form of a combination. Rhodiola rosea exosomes also have a certain effect of increasing the moisture content of volunteers' skin, but their effect is not as good as the small molecule collagen peptide HPTAE (His-Pro-Thr-Ala-Glu) of the present invention, and the effect is even worse when used in combination with the small molecule collagen peptide HPTAE (His-Pro-Thr-Ala-Glu).

[0101] In addition, the comparative small molecule collagen peptide HPSAE (His-Pro-Ser-Ala-Glu) or calendula exosomes alone could not improve the skin moisture content of volunteers.

[0102] However, the combination of the small molecule collagen peptide HPTAE (His-Pro-Thr-Ala-Glu) of the present invention and calendula exosomes can significantly improve the skin moisture content of volunteers, and its effect is better than the effect produced by the combination of the small molecule collagen peptide of the present invention and Rhodiola rosea exosomes, which have the effect of improving skin moisture content. This shows that the combination of a specific small molecule collagen peptide and a specific type of plant-derived exosomes has an unexpectedly significant synergistic effect in improving the skin moisture content of volunteers and enhancing skin moisturizing.

[0103] Table 2: Effect of the composition with skin anti-aging effect of the present invention on skin moisturizing

[0104] Group Skin water content increase rate (%) Cream base 6.25 Example 1 group 50.45 Example 2 group 47.38 Example 3 group 48.53 Comparative Example 1 40.54 Comparative Example 2 10.37 Comparative Example 3 9.22 Comparative Example 4 19.96 Comparative Example 5 40.14 Comparative Example 6 9.83 Comparative Example 7 14.26

[0105] Effect Example 3: Anti-wrinkle and firming effect test of the composition with skin anti-aging effect of the present invention

[0106] 88 women aged 35-65 were selected as subjects and divided into 11 groups of 8 each. Before the test, the subjects washed their faces and stayed in a constant temperature and humidity environment (temperature 21-23°C, humidity 55-65%) for 30 minutes. Their faces were photographed using the VISA-CR for initial data. The subjects then applied the corresponding facial cream (the same cream used in Effectiveness Example 2) to their faces once daily for 30 consecutive days. On days 0 and 30, facial wrinkle measurements were performed using the VISA-CR after washing their faces, with these values recorded as D0 and D30, respectively. The D0 and D30 values for each group were the averages of these values for the group. Instrument software calculated the improvement in facial wrinkles before and after use, with lower scores indicating more wrinkles.

[0107] The test results are shown in Table 3 below. As can be seen from the data in Table 3, the skin anti-wrinkle effect of conventional cosmetic base ingredients used to prepare facial creams when used alone is poor, with essentially no wrinkle-removing effect. The composition with skin anti-aging effects of the present invention and the small molecule collagen peptide HPTAE (His-Pro-Thr-Ala-Glu) have significant anti-wrinkle effects when used alone, and the effect is even better when used in the form of a composition. Rhodiola rosea exosomes have a slight anti-wrinkle effect, but their effect is not as good as the small molecule collagen peptide HPTAE (His-Pro-Thr-Ala-Glu) of the present invention, and when used in combination with the small molecule collagen peptide HPTAE (His-Pro-Thr-Ala-Glu), the effect is even worse. In addition, the comparative small molecule collagen peptide HPSAE (His-Pro-Ser-Ala-Glu) or calendula exosomes have no anti-wrinkle effect when used alone. However, the combination of the small molecule collagen peptide HPTAE (His-Pro-Thr-Ala-Glu) of the present invention and marigold exosomes can significantly enhance the anti-wrinkle effect of small molecule collagen, and its effect is better than the effect produced by the combination of the small molecule collagen peptide of the present invention and Rhodiola rosea exosomes, which have a slight anti-wrinkle effect. This shows that the combination of specific small molecule collagen peptides and specific types of plant-derived exosomes has an unexpectedly significant synergistic effect in anti-wrinkle and anti-aging.

[0108] Table 3: Anti-wrinkle effect test results of the composition with skin anti-aging effect of the present invention

[0109]

[0110]

[0111] In summary, the experimental results show that in the composition of the present invention, by combining small molecule collagen peptides and calendula exosomes in a specific dosage ratio, the composition of the present invention has synergistic moisturizing and skin barrier repair as well as anti-wrinkle and firming effects. When applied to cosmetics, it can promote the reconstruction of the skin's stratum corneum and barrier function, improve the skin's moisturizing and water resistance, reduce skin wrinkles and make the skin firmer, and provide an excellent user experience.

[0112] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A composition having skin anti-aging effects, characterized in that: The composition comprises small molecule collagen peptides and marigold exosomes, and the weight ratio of the small molecule collagen peptides to the marigold exosomes is 1-10:10-1.

2. The composition according to claim 1, wherein: The weight ratio of the small molecule collagen peptide to the marigold exosomes is 1-8:8-1.

3. The composition according to claim 1, wherein: The small molecule collagen peptide is separated and identified from deep-sea cod skin enzymatic hydrolysate, and the amino acid sequence of the small molecule collagen peptide is HPTAE (His-Pro-Thr-Ala-Glu).

4. The composition according to claim 3, characterized in that: The small molecule collagen peptide is prepared by solid phase synthesis.

5. The composition according to claim 1, wherein: The marigold exosomes have a double membrane structure under a transmission electron microscope, are mostly in the shape of a concave round cake, and have a particle size of 50-300 nm.

6. The composition according to claim 5, characterized in that: The preparation method of marigold exosomes comprises the following steps: (1) Fresh marigolds washed with pure water were soaked in an appropriate amount of PBS, and after being fully soaked, they were crushed in a wall-breaking machine, and filtered through a gauze to obtain filtrate a; (2) centrifuging the filtrate a at 1,000 g for 10-15 min to obtain supernatant liquid a, and centrifuging the supernatant liquid a at 3,000 g for 30-40 min to obtain supernatant liquid b; (3) Supernatant b was concentrated by centrifugation at 3,500 g for 30-40 min using a 100 kDa ultrafiltration tube to obtain supernatant concentrate c, and supernatant concentrate c was centrifuged at 10,000 g for 30-40 min to obtain supernatant concentrate d; (4) Filter the supernatant concentrate d through a 0.45 μm filter membrane to obtain filtrate b; (5) The filtrate b is subjected to ultrahigh-speed centrifugation at 100,000 to 120,000 g for 60 to 90 minutes to obtain a precipitate, thereby obtaining the marigold exosomes.

7. Use of the composition with skin anti-aging effect according to any one of claims 1 to 6 in the preparation of cosmetics with moisturizing and skin barrier repairing, anti-wrinkle and firming effects.

8. The use according to claim 7, characterized in that: The cosmetics are toner, lotion, essence, cream, ointment, facial mask or freeze-dried powder.

9. The use according to claim 7, characterized in that: The cosmetic has a protective effect on UV-induced cell damage, has antioxidant activity, can repair damaged cells, protect the skin barrier, and delay skin aging.

10. The use according to claim 7, characterized in that: Calculated by mass percentage, the content of the composition in the cosmetic is 1%-5%.

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