A composition containing hydroxypinazone 9-cis-retinate exosomes and its application

Abstract

This invention discloses a composition containing exosomes loaded with hydroxypinazone 9-cis-retinate and its application, belonging to the technical field of cosmetic raw materials. The core components of this composition are agarwood exosomes loaded with hydroxypinazone 9-cis-retinate and silk protein. This composition achieves a synergistic effect through the highly efficient delivery characteristics of agarwood exosomes and the film-forming and moisturizing properties of silk protein. It solves the problems of instability, poor skin permeability, and strong irritation associated with traditional retinol-based ingredients, and overcomes the shortcomings of existing exosome delivery technologies, such as poor compatibility of active ingredients, easy inactivation of exosomes, and difficulty in staying on the skin surface. It achieves deep penetration, sustained release, and targeted action of hydroxypinazone 9-cis-retinate, significantly improving firming, anti-wrinkle, whitening, and spot-fading effects while reducing skin irritation. A firming and anti-wrinkle serum prepared based on this composition has been experimentally verified to have excellent skin-improving effects.

Description

Technical Field

[0001] This invention belongs to the technical field of cosmetic raw materials, specifically relating to a composition containing hydroxypinazone 9-cis-retinate exosomes and its application. Background Technology

[0002] Retinic acid and its derivatives (such as retinol and retinoic acid esters) are widely recognized as among the most effective anti-aging ingredients, capable of promoting collagen production, accelerating keratinocyte renewal, and reducing wrinkles and age spots. 9-cis-retinoic acid ester is an isomer of retinoic acid ester and possesses unique biological activity.

[0003] However, traditional retinol-based ingredients have obvious limitations: 1) They are chemically unstable and easily oxidized and deactivated; 2) They have poor skin permeability and are difficult to effectively reach target cells in the dermis; 3) They are highly irritating and can easily cause adverse reactions such as dry skin, redness, and peeling, which limits their widespread application.

[0004] To overcome these challenges, the industry is constantly exploring new delivery technologies. Exosomes, as natural nanoscale lipid vesicles secreted by cells, are considered highly promising drug and active ingredient delivery carriers due to their excellent biocompatibility, low immunogenicity, and efficient transmembrane transport capabilities. In recent years, the encapsulation of active ingredients using exosomes has become a research hotspot in the cosmetics field.

[0005] The prior art CN118845493A discloses a scheme for co-encapsulating Proxylane and retinol derivatives with milk exosomes, which confirms the feasibility of exosomes as carriers of active ingredients in cosmetics. However, this scheme has significant limitations: First, the combination of multiple active ingredients with different physicochemical properties presents compatibility issues, making it difficult to precisely control the drug loading and release behavior of each ingredient; Second, direct application of exosomes in this scheme is prone to drying and inactivation, and it is difficult to effectively remain on the skin surface, affecting its final efficacy. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide an exosome-silk protein composition containing hydroxypinazone 9-cis-retinate loaded with hydroxypinazone. This composition achieves deep penetration, sustained release and targeted action of hydroxypinazone 9-cis-retinate through the efficient delivery of exosomes and the film-forming and moisturizing synergistic effect of silk protein, significantly enhancing its anti-aging and whitening effects while greatly reducing skin irritation.

[0007] To achieve the above objectives, the present invention discloses the following technical solutions:

[0008] In a first aspect, the present invention provides a composition containing hydroxypinazone 9-cis-retinoate-loaded exosomes, said composition containing hydroxypinazone 9-cis-retinoate-loaded exosomes and filoin.

[0009] Preferably, the concentration of the hydroxypinazone-9-cis-retinate-loaded exosomes is (1-10)×10⁻⁶. 9 PCS / mL;

[0010] The mass ratio of hydroxypinazone-9-cis-retinoate-loaded exosomes and filoin in the composition is (0.02-0.3):1.

[0011] Preferably, the silk protein is a water-soluble silk protein.

[0012] More preferably, the method for preparing the hydroxypinazone-9-cis-retinate-loaded exosomes includes the following steps:

[0013] 1-1. After homogenizing the agarwood leaves by breaking the cell wall, filter to remove coarse impurities. The resulting filtrate is separated by differential and ultracentrifugation to obtain a crude extract rich in agarwood exosomes. Then, it is concentrated by cross-flow filtration to obtain an agarwood exosome suspension.

[0014] 1-2. The agarwood exosome suspension was mixed with a solution containing hydroxypinazone 9-cis-retinate and incubated under light-protected conditions to load hydroxypinazone 9-cis-retinate into the agarwood exosomes. The mixture was then concentrated by cross-flow filtration to obtain the hydroxypinazone 9-cis-retinate loaded exosomes.

[0015] More preferably, the differential and ultracentrifugation involves centrifuging the filtrate at a low speed of 3000-5000×g for 20-30 min, then centrifuging at a medium speed of 10000-15000×g for 30-45 min, and finally centrifuging at an ultracentrifugation of 100000-120000×g for 70-120 min, and collecting the precipitate.

[0016] More preferably, the diameter of the filter pores for cross-flow filtration concentration in step 1-1 is 100-500 nm.

[0017] More preferably, the diameter of the filter pores for cross-flow filtration concentration in steps 1-2 is 200-300 nm.

[0018] More preferably, the method for preparing the solution containing hydroxypinazone 9-cis-retinate in steps 1-2 includes the following steps:

[0019] Hydroxypinazone 9-cis-retinate was dissolved in anhydrous ethanol, and then PBS solution was added and homogenized. The concentration of ethanol in the system was ≤5v / v%, and the solution containing hydroxypinazone 9-cis-retinate was obtained.

[0020] The co-incubation temperature is 25-30℃, and the co-incubation time is 30-60 min. The co-incubation is carried out in the dark and under nitrogen or argon protection. After the co-incubation is completed, the mixture is concentrated by cross-flow filtration to obtain the loaded hydroxypinazone 9-cis-retinate exosomes.

[0021] Secondly, the composition described in the first aspect of the present invention is used in the preparation of skin care products having firming, anti-wrinkle, whitening and / or spot-fading effects.

[0022] Thirdly, the present invention provides a firming and anti-wrinkle serum, which, by weight percentage, comprises the following components:

[0023] 0.01-0.05% silken protein, 0.001-0.01% exosome suspension loaded with hydroxypinazone 9-cis-retinate, 1-10% glycerol, 1-10% butylene glycol, 0.1-0.6% p-hydroxyacetophenone, 0.2-0.6% hydroxyethyl cellulose, and the balance deionized water.

[0024] Fourthly, the present invention provides a method for preparing the serum described in the third aspect, the method comprising the following steps:

[0025] 2-1. First, dissolve the silk core protein in deionized water, then add the exosome suspension loaded with hydroxypinazone 9-cis-retinoate, and stir and disperse evenly at 1000-2000 r / min to obtain solution A;

[0026] 2-2. Add p-hydroxyacetophenone and hydroxyethyl cellulose to glycerol and butanediol at a temperature not exceeding 60°C, stir and disperse evenly at 1500-3000 r / min to obtain solution B, and cool to 30°C or below;

[0027] 2-3. Stir and disperse solution B and solution A at 1000-2000 r / min at a temperature not exceeding 30°C to obtain the essence.

[0028] In this invention:

[0029] Hydroxypinazone 9-cis-retinoic acid (9-CIS HPR) is an effective anti-aging ingredient that promotes collagen production, accelerates keratinocyte renewal, and reduces wrinkles and age spots. 9-cis-retinoic acid is an isomer of retinoic acid and possesses unique biological activity. Hydroxypinazone 9-cis-retinoic acid is a novel, relatively stable retinoic acid precursor that can be converted into the active form of 9-cis-retinoic acid within the skin to exert its effects.

[0030] Exosomes, as a naturally derived nanovesicle, are considered a promising next-generation delivery system due to their excellent biocompatibility, low immunogenicity, and inherent intercellular communication and material delivery capabilities.

[0031] Silk protein is a natural high-molecular-weight protein extracted from silk. It has excellent film-forming properties, moisturizing properties and biocompatibility. At the same time, it is rich in amino acids and peptides. In cosmetics, it can form a breathable protective film on the skin surface, lock in moisture and smooth the skin.

[0032] This invention innovatively loads hydroxypinazone 9-cis-retinate into agarwood exosomes, reducing its irritation to the skin while better leveraging the whitening and anti-aging effects of hydroxypinazone 9-cis-retinate. The exosomes provide an effective delivery and stabilizing shell for the active substances, and the fibroin provides the necessary support for the effective adhesion and retention of exosomes on the skin surface, improving the duration of action of exosomes and solving the problem of effective retention of exosomes on the skin surface.

[0033] The beneficial effects of this invention are:

[0034] 1. In this invention, agarwood exosomes, as natural nano-sized lipid vesicles, provide a stable "protective shell" for 9-CIS HPR with their good biocompatibility, low immunogenicity and efficient transmembrane transport ability, reducing its oxidative loss during storage and use; at the same time, exosomes can carry 9-CIS HPR to penetrate the stratum corneum of the skin and reach target cells such as fibroblasts in the dermis, thereby improving the utilization rate of active ingredients.

[0035] 2. Water-soluble silk protein can form a breathable protective film on the skin surface, which locks in skin moisture and plays a moisturizing role. On the other hand, it provides a stable attachment carrier for agarwood exosomes, reducing the risk of exosomes becoming inactive due to dryness, prolonging their residence time on the skin surface, ensuring the continuous release and efficacy of 9-CIS HPR, and achieving the synergistic effect of "efficient delivery of exosomes + retention and moisturizing with silk protein". Attached Figure Description

[0036] Figure 1 This is a particle size distribution diagram of the exosome suspension before concentration and filtration.

[0037] Figure 2 This is a transmission electron microscope image of the concentrated exosome suspension. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. However, the following embodiments are merely preferred embodiments of this invention and not all of them. For clarity, not all features of the actual embodiments are described.

[0039] Based on the embodiments described in the implementation plan, other embodiments obtained by those skilled in the art without creative effort are all within the scope of protection of this invention.

[0040] In this invention:

[0041] Silk core protein: Water-soluble silk core protein, purchased from Senset (Suzhou) Biotechnology Co., Ltd.;

[0042] Hydroxypinazone 9-cis-retinate (9-CIS HPR): prepared according to Example 1 in patent CN116947723A;

[0043] Agarwood leaves: The leaves of Aquilaria sinensis, a species of agarwood in the Thymelaeaceae family, are commercially available.

[0044] Preparation methods of plant exosomes

[0045] Step 1-1: Place fresh agarwood leaves in a high-speed blender, add PBS buffer at a material-to-liquid ratio of 1:1 g / mL, start the blender to break down the leaves, and obtain agarwood homogenate. Pass the homogenate through a 200-mesh sieve for preliminary filtration to remove large plant fibers, tissue fragments and other impurities, and obtain a clear filtrate.

[0046] Steps 1-2: Centrifuge the filtrate at 4000×g for 25 min to remove large cell debris and undisturbed tissue. Then centrifuge at 15000×g for 45 min to further remove smaller organelles and larger vesicles. Finally, ultracentrifuge at 120000×g for 80 min. After centrifugation, the exosomes will precipitate at the bottom of the tube. Discard the supernatant and resuspend the precipitate in pre-cooled PBS buffer to obtain the suspension. Take the required amount of suspension for analysis, dilute it a certain factor, and then perform particle size distribution analysis. The results are as follows: Figure 1 As shown, it is then concentrated by cross-flow filtration with a pore diameter of 100-500 nm to obtain an agarwood exosome suspension.

[0047] The transmission electron microscopy image of the agarwood exosomes is shown below. Figure 2 As shown.

[0048] Preparation of exosomes loaded with hydroxypinazone 9-cis-retinate

[0049] Step 2-1: Dissolve 9-CIS HPR in a small amount of anhydrous ethanol to prepare a high-concentration stock solution. Add the stock solution to PBS solution and homogenize it. Control the volume fraction of ethanol in the system to not exceed 5% (v / v) to obtain a 9-CIS HPR working solution with a concentration of 2 mg / mL.

[0050] Step 2-2: Mix the agarwood exosome suspension with the 9-CIS HPR working solution at a volume ratio of 1:1, and gently stir and incubate at 25°C for 60 min under light-protected and nitrogen-protected conditions to allow the 9-CIS HPR to embed into the exosome bilayer membrane;

[0051] Steps 2-3: The incubated mixture is concentrated by cross-flow filtration with a pore size of 200-300 nm to finally obtain 9-CIS HPR-loaded exosomes.

[0052] Performance testing

[0053] 1. Reactive Oxygen Species Inhibition Test

[0054] 1.1 Experimental Methods

[0055] Refer to T / SHRH 032-2020 "Test Method for Firming and Anti-wrinkle Efficacy of Cosmetics - In Vitro Test Method for Inhibition of Reactive Oxygen Species (ROS) in Keratinocytes".

[0056] 1.2 Experimental Grouping

[0057] A positive control group, an agarwood exosome treatment group, a 9-CIS HPR treatment group, a milk exosome treatment group, a milk exosome treatment group loaded with 9-CIS HPR, and an agarwood exosome treatment group loaded with 9-CIS HPR were set up respectively; the stimulant was hydrogen peroxide, and the induction dose was 250 μmol / L.

[0058] The settings for the above groups are as follows:

[0059] Agarwood exosome treatment group: Agarwood exosome suspension was diluted to 5.0 × 10⁻⁶. 7 PCS / mL, 2v / v% diluted agarwood exosome suspension was added to the complete culture medium to treat the cells, and this group was called the agarwood exosome group;

[0060] 9-CIS HPR treatment group: 9-CIS HPR was diluted with anhydrous ethanol to a concentration of 1 mg / mL, and 2 v / v% of the diluted 9-CIS HPR solution was added to the complete culture medium to treat the cells. This group was designated as the 9-CIS HPR treatment group.

[0061] Milk exosome treatment group: A certain amount of commercially available milk exosomes (Yumeibo Biotechnology) was diluted with PBS buffer to a concentration of 5.0 × 10⁻⁶. 7 PCS / mL, 2v / v% diluted milk exosome suspension was added to the complete culture medium to treat the cells, and this group was called the milk exosome treatment group.

[0062] Milk exosomes loaded with 9-CIS HPR: Following steps 2-1 to 2-3, the exosomes were replaced with milk exosomes (Yumeibo Biotechnology). The resulting milk exosomes loaded with 9-CIS HPR were diluted with PBS buffer to 5.0 × 10⁻⁶. 7 PCS / mL, milk exosomes loaded with 9-CIS HPR diluted to 2v / v% were added to the complete culture medium to treat the cells, and this group was designated as the milk exosomes loaded with 9-CIS HPR treatment group.

[0063] Agarwood exosomes loaded with 9-CIS HPR: The exosomes loaded with 9-CISHPR prepared in steps 2-1 to 2-3 above were diluted to 5.0 × 10⁻⁶. 7 PCS / mL, 2v / v% diluted exosomes loaded with 9-CIS HPR were added to the complete culture medium to treat the cells, and this group was called the exosome treatment group loaded with 9-CIS HPR.

[0064] Positive control group: Vitamin E was diluted to a solution of 0.05 g / mL with anhydrous ethanol, and then diluted to 0.05% with complete culture medium at a volume ratio of 1:100.

[0065] The results are shown in Table 1.

[0066] 1.3 Experimental Results

[0067] Table 1. Results of ROS inhibition rate

[0068] Group Inhibition rate / % Positive control group 86.17 Agarwood exosome treatment group 12.82 9-CIS HPR Processing Group 31.23 Milk exosome treatment group 10.94 Milk exosomes loaded with 9-CIS HPR 70.32 Exosome treatment group loaded with 9-CIS HPR 82.63

[0069] 1.4 Results Analysis

[0070] The results showed that the ROS inhibition rate of the agarwood exosome group alone was 12.82%, and the ROS inhibition rate of the milk exosome group alone was 10.94%, which were relatively poor, indicating that the ROS scavenging ability of exosomes alone was poor. The inhibition rate of the 9-CIS HPR group alone was 31.23%, indicating that 9-CIS HPR has certain antioxidant activity.

[0071] The inhibition rate of milk exosomes loaded with 9-CIS HPR was 70.32%, which was significantly higher than that of the 9-CIS HPR alone group and the milk exosome alone group, demonstrating that the carrier effect of exosomes can enhance the antioxidant efficacy of 9-CIS HPR.

[0072] The inhibition rate of agarwood exosomes loaded with 9-CIS HPR was 82.63%, which was closer to the positive control group and superior to the milk exosomes loaded with 9-CIS HPR. This indicates that agarwood exosomes, as a carrier, are more efficient than milk exosomes in protecting the stability of 9-CIS HPR and promoting its entry into cells to exert its effects.

[0073] The 9-CIS HPR agarwood exosomes provided by this invention can effectively reduce oxidative stress levels and achieve antioxidant and anti-aging effects.

[0074] 2. Effect of Type I Collagen Content Test

[0075] 2.1 Experimental Methods

[0076] Refer to T / SHRH 031-2020 "Testing of Firming and Anti-wrinkle Efficacy of Cosmetics - Determination of Type I Collagen Content in In Vitro Fibroblasts".

[0077] Dilute the cells with cell culture medium to the seeding density (confluence reaches 45%–60% 24 hours after seeding), and seed them into 96-well plates, with 200 μL per well. After seeding, incubate in a CO2 incubator for 24 h ± 2 h.

[0078] Discard the culture medium in the 96-well plate and proceed with the drug administration. Add culture medium containing the test substance to the test wells, culture medium containing the positive control to the positive control wells, and normal cell culture medium to the blank / solvent control wells, 200 μL per well. After drug administration, incubate the 96-well plate in a CO2 incubator for 24 h ± 2 h. After incubation, collect 200 μL of cell culture supernatant from each well into a 1.5 mL sterile centrifuge tube and store it at -80°C for later analysis. The type I collagen content in the supernatant was determined according to the instructions for use of the Human Type I Collagen Enzyme-Linked Immunosorbent Assay Kit.

[0079] 2.2 Test substance

[0080] Agarwood exosome treatment group: Agarwood exosome suspension was diluted to 5.0 × 10⁻⁶. 7 PCS / mL, 2v / v% diluted agarwood exosome suspension was added to the cell culture medium to treat the cells, and this group was called the agarwood exosome group;

[0081] 9-CIS HPR treatment group: 9-CIS HPR was diluted with anhydrous ethanol to a concentration of 1 mg / mL, and 2 v / v% of the diluted 9-CIS HPR solution was added to the cell culture medium to treat the cells. This group was designated as the 9-CIS HPR treatment group.

[0082] Milk exosome treatment group: A certain amount of commercially available milk exosomes (Yumeibo Biotechnology) was diluted with PBS buffer to a concentration of 5.0 × 10⁻⁶. 7 PCS / mL, 2v / v% diluted milk exosome suspension was added to the cell culture medium to treat the cells, and this group was called the milk exosome treatment group;

[0083] Milk exosomes loaded with 9-CIS HPR: Following steps 2-1 to 2-3, the exosomes were replaced with milk exosomes (Yumeibo Biotechnology). The resulting milk exosomes loaded with 9-CIS HPR were diluted with PBS buffer to 5.0 × 10⁻⁶. 7 PCS / mL, milk exosomes loaded with 9-CIS HPR diluted 2v / v% were added to the cell culture medium to treat the cells, and this group was called the milk exosomes loaded with 9-CIS HPR treatment group.

[0084] 9-CIS HPR-loaded exosome treatment group: The 9-CIS HPR-loaded exosomes prepared in steps 2-1 to 2-3 above were diluted to 5.0 × 10⁻⁶. 7 PCS / mL, 2v / v% diluted exosomes loaded with 9-CIS HPR were added to the cell culture medium to treat the cells, and this group was called the exosomes loaded with 9-CIS HPR treatment group.

[0085] Positive control group: 100 ng / mL TGFβ1 working solution (prepared according to Appendix B of T / SHRH 031-2020).

[0086] 2.3 Experimental Results

[0087] Table 2. Results of COL-I content

[0088] Group <![CDATA[COL-I content / ng·mL -1 > Upward adjustment rate / % Blank control group 103.44 / Positive control group 136.74 32.19% Agarwood exosome treatment group 113.85 10.06% 9-CIS HPR Processing Group 134.38 29.91% Milk exosome treatment group 116.76 12.88% Milk exosomes loaded with 9-CIS HPR 148.12 43.19% Exosome treatment group loaded with 9-CIS HPR 169.08 63.46%

[0089] 2.4 Results Analysis

[0090] The blank control group had a COL-I content of 103.44 ng / mL, which served as the baseline reference; the positive control group (TGFβ1) had an upregulation rate of 32.19%, verifying the effectiveness of the experimental method.

[0091] In the exosome-only group, agarwood exosomes were upregulated by 10.06% and milk exosomes by 12.88%, which had a certain promoting effect on collagen synthesis, but the effect was limited; the upregulation rate in the 9-CIS HPR-only group was 29.91%, indicating that 9-CIS HPR can stimulate fibroblasts to synthesize collagen.

[0092] The upregulation rate of milk exosomes loaded with 9-CIS HPR was 43.19%, which was better than that of the 9-CIS HPR alone group and the milk exosome alone group, indicating that the exosome carrier can enhance the promoting effect of 9-CIS HPR on collagen synthesis.

[0093] The upregulation rate of agarwood exosomes loaded with 9-CIS HPR was 63.46%, which was significantly higher than all other groups, demonstrating that agarwood exosomes loaded with 9-CIS HPR more effectively activated intracellular collagen synthesis-related pathways and significantly increased COL-I content.

[0094] Preparation of application examples

[0095] Step 3-1: Accurately weigh the raw materials according to the percentage of mass in Table 3. First, dissolve the silk protein in deionized water, then add water with a concentration of 5×10⁻⁶. 9 PCS / mL of exosome suspension loaded with 9-CIS HPR was added, and the mixture was stirred and dispersed evenly at 1500 r / min to obtain solution A;

[0096] Step 3-2: Add p-hydroxyacetophenone and hydroxyethyl cellulose to glycerol and butanediol at 50℃, stir and disperse evenly at 2000 r / min to obtain solution B, and cool to 30℃.

[0097] Step 3-3: Stir and disperse solution B and solution A at 1500 r / min at 30℃ to obtain the essence.

[0098] Table 3. Raw material mass percentage

[0099] Raw material name Application Example 1 Application Example 2 Application Example 3 Comparative Example 1 Comparative Example 2 Silk Heart Protein 0.05 0.05 0.033 0.05 / Exosome suspension loaded with 9-CIS HPR 0.001 0.005 0.01 / 0.005 glycerin 2 2 2 2 2 Butylene glycol 1 1 1 1 1 p-Hydroxyacetophenone 0.5 0.5 0.5 0.5 0.5 Hydroxyethyl cellulose 0.3 0.3 0.3 0.3 0.3 Deionized water Add to 100 Add to 100 Add to 100 Add to 100 Add to 100

[0100] Note: " / " in the table indicates no addition.

[0101] 3. Human efficacy test

[0102] 3.1 Test Method

[0103] Fifty female volunteers with wrinkles and healthy, undamaged skin, with an average age of 45±4 years, were randomly divided into 5 groups of 10 each, using the application examples 1-3 and comparative examples 1-2 described above. Each subject applied 2g of the serum to their face twice daily, morning and evening, for 30 consecutive days.

[0104] During the experiment, subjects were not allowed to apply any other cosmetics to the test areas. Data were collected using a Cutometer MPA580 skin elastometer on day 0 and day 30, with the test area being the cheekbone. Measurements were taken three times on the same test area, and the average value was recorded.

[0105] The test parameter is R2 (the ratio of skin elasticity Ua without negative pressure to maximum stretch Uf with negative pressure). The closer R2 is to 1, the better the skin elasticity. R2 change rate = R2 value on day 30 - R2 value on day 0 / R2 value on day 0.

[0106] 3.2 Test Results

[0107] Skin elasticity test results

[0108] Group Initial R2 value R2 value on day 30 R² rate of change / % Application Example 1 0.499 0.582 16.63% Application Example 2 0.495 0.601 21.41% Application Example 3 0.493 0.602 22.11% Comparative Example 1 0.498 0.519 4.22% Comparative Example 2 0.492 0.523 6.30%

[0109] 3.3 Results Analysis

[0110] Comparative Example 1, containing filamentin but without exosomes loaded with 9-CIS HPR, showed an R² change rate of only 4.22%. Comparative Example 2, containing exosomes loaded with 9-CIS HPR but without filamentin, showed an R² change rate of 6.30%. Both showed poor results, indicating that filamentin alone can only provide basic moisturizing effects and cannot significantly improve skin elasticity. Exosomes loaded with 9-CIS HPR alone have difficulty staying on the skin surface, preventing the active ingredients from exerting their effects continuously, resulting in limited efficacy.

[0111] The R² change rate of Application Examples 1-3 was 16.63%-22.11%, significantly higher than that of the two comparative examples, demonstrating that there is a synergistic effect between filamentin and agarwood exosomes loaded with 9-CIS HPR: the membrane-forming retention effect of filamentin ensures the continuous release of 9-CIS HPR by exosomes, and the efficient delivery of 9-CIS HPR to target cells by agarwood exosomes promotes collagen synthesis. The combined effect of the two significantly improves skin elasticity.

[0112] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A composition containing hydroxypinazone 9-cis-retinate exosomes, characterized in that, The composition contains hydroxypinazone 9-cis-retinate-loaded exosomes and filoin; The method for preparing the loaded hydroxypinazone 9-cis-retinate exosomes includes the following steps: 1-1. After homogenizing the agarwood leaves by breaking the cell wall, filter to remove coarse impurities. The resulting filtrate is separated by differential and ultracentrifugation to obtain a crude extract rich in agarwood exosomes. Then, it is concentrated by cross-flow filtration to obtain an agarwood exosome suspension. 1-2. The agarwood exosome suspension was mixed with a solution containing hydroxypinazone 9-cis-retinate and incubated under light-protected conditions to load hydroxypinazone 9-cis-retinate into the agarwood exosomes. The mixture was then concentrated by cross-flow filtration to obtain the hydroxypinazone 9-cis-retinate loaded exosomes. The concentration of the loaded hydroxypinazone 9-cis-retinate exosomes was (1-10)×10⁻⁶. 9 PCS / mL; The mass ratio of hydroxypinazone-9-cis-retinoate-loaded exosomes to filoin in the composition is (0.02-0.3):1; The silk core protein is water-soluble silk core protein; The preparation method of the solution containing hydroxypinazone 9-cis-retinate in steps 1-2 includes the following steps: Hydroxypinazone 9-cis-retinate was dissolved in anhydrous ethanol, and then PBS solution was added and homogenized. The concentration of ethanol in the system was ≤5v / v%, and the solution containing hydroxypinazone 9-cis-retinate was obtained. The co-incubation temperature is 25-30℃, and the co-incubation time is 30-60 min. The co-incubation is carried out in the dark and under nitrogen or argon protection. After the co-incubation is completed, the mixture is concentrated by cross-flow filtration to obtain the loaded hydroxypinazone 9-cis-retinate exosomes.

2. The composition according to claim 1, characterized in that, The differential and ultracentrifugation process involves centrifuging the filtrate at a low speed of 3000-5000×g for 20-30 min, then centrifuging at a medium speed of 10000-15000×g for 30-45 min, and finally centrifuging at an ultracentrifugation of 100000-120000×g for 70-120 min, and collecting the precipitate.

3. The composition according to claim 1, characterized in that, The diameter of the filter pores used for cross-flow filtration concentration in step 1-1 is 100-500 nm.

4. The composition according to claim 1, characterized in that, The diameter of the filter pores used for cross-flow filtration concentration in steps 1-2 is 200-300 nm.

5. The use of the composition according to any one of claims 1-4 in the preparation of skin care products having firming, anti-wrinkle and / or whitening and spot-fading effects.

6. A firming and anti-wrinkle serum, characterized in that, The serum comprises the following components by weight percentage: 0.01-0.05% silken protein, 0.001-0.01% exosome suspension loaded with hydroxypinazone 9-cis-retinate, 1-10% glycerol, 1-10% butylene glycol, 0.1-0.6% p-hydroxyacetophenone, 0.2-0.6% hydroxyethyl cellulose, and the balance deionized water.

7. The method for preparing the essence according to claim 6, characterized in that, The preparation method includes the following steps: 2-1. First, dissolve the silk core protein in deionized water, then add the exosome suspension loaded with hydroxypinazone 9-cis-retinoate, and stir and disperse evenly at 1000-2000 r / min to obtain solution A; 2-2. Add p-hydroxyacetophenone and hydroxyethyl cellulose to glycerol and butanediol at a temperature not exceeding 60°C, stir and disperse evenly at 1500-3000 r / min to obtain solution B, and cool to 30°C or below; 2-3. Stir and disperse solution B and solution A at 1000-2000 r / min at a temperature not exceeding 30°C to obtain the essence.

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