Salidroside composition with core-shell-shell structure and preparation method and use thereof

Abstract

The present application provides a rhodiolide composition with a core-shell-shell structure, a preparation method thereof comprising the steps of: 1) EGCG inclusion: adding beta-cyclodextrin and EGCG into water, ultrasonic, forming an inclusion compound; 2) liposome assembly: weighing soybean phospholipid and cholesterol, dissolving in chloroform to form a uniform solution; weighing rhodiolide, dissolving in the uniform solution, rotary evaporation to remove chloroform, then adding the inclusion compound and PBS buffer into the container, hydrating, forming a liposome wrapping rhodiolide and inclusion compound; 3) shell loading: weighing collagen, mixing with chitosan solution uniformly to form a complex; adding the complex into the liposome, the complex is coated on the outer layer of the liposome, centrifugal purification, obtaining a rhodiolide composition with a core-shell-shell structure. The present application belongs to the technical field of cosmetic raw materials, and the rhodiolide composition provided has the advantages of high stability, good transdermal effect, and significant anti-aging effect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cosmetic raw materials, and particularly relates to a rhodiolide composition with a core-shell-shell structure and a preparation method and application thereof. BACKGROUND

[0002] In the field of cosmetic raw materials, rhodiolide, collagen and epigallocatechin gallate (EGCG) are highly concerned due to their good anti-aging effects and play an important role in the field of cosmetics. Rhodiolide has the effects of antioxidant and cell metabolism regulation; collagen can maintain the elasticity and firmness of the skin; and EGCG is a highly efficient antioxidant that can scavenge free radicals and inhibit inflammatory reactions. However, rhodiolide has poor long-term stability in aqueous solution and is also susceptible to light and oxidation. The molecular weight of recombinant collagen is usually 30-500 kDa, and according to Franz diffusion cell data, its transdermal rate is less than 5%. The large molecular weight of collagen makes it difficult to penetrate the stratum corneum barrier of the skin, and it cannot effectively penetrate into the deep layer of the skin, resulting in a significant reduction in anti-aging effect. EGCG is sensitive to light, and its half-life is less than 24 h. Through high performance liquid chromatography (HPLC) detection, it is found that the degradation rate of EGCG after light exposure is as high as 80%, resulting in a large loss of active ingredients and making it difficult to fully exert its anti-aging effect.

[0003] In order to improve the stability of rhodiolide, the existing technology mainly prepares rhodiolide into liposomes. Chinese patent application CN 113041169 A discloses a rhodiolide liposome lyophilized powder, which is prepared from rhodiolide, egg yolk lecithin and cholesterol as raw materials, and has the advantages of good stability. Since EGCG has a negative charge and collagen has a positive charge at pH < 5, direct mixing of EGCG and collagen will result in electrostatic attraction and precipitation. In order to overcome the problems of collagen and EGCG in application, Chinese patent application CN 117137818 A discloses an EGCG and recombinant collagen self-assembled microsphere, which realizes the co-use of EGCG and recombinant collagen by using self-assembly technology, i.e. EGCG and proline and hydroxyproline of recombinant collagen COL are combined through intermolecular hydrogen bond and π bond non-covalent bond to form nanometer microspheres, which can improve the permeability of collagen and inhibit the oxidation of EGCG, and reduce the cytotoxicity of EGCG. Since rhodiolide has fat solubility and has compatibility problems with water-soluble collagen, phase separation is easy to occur after mixing, which affects the stability and uniformity of the system and leads to unstable product quality. It can be seen that rhodiolide, collagen and EGCG are difficult to be combined and applied by simple mixing.

[0004] Therefore, it is of great significance to provide a salidroside composition with a core-shell-shell structure and a preparation method and application thereof. SUMMARY

[0005] To solve the problems in the prior art, the present application provides a salidroside composition with a core-shell-shell structure (hereinafter referred to as the inventive composition). By designing a core-shell-shell three-layer carrier structure, β-cyclodextrin, liposomes and chitosan-collagen complex are used to sequentially perform inclusion, assembly and loading, respectively, to solve the charge conflict between EGCG and collagen and the phase separation problem of salidroside and collagen. At the same time, EGCG is effectively protected from light degradation, the transdermal rate of active ingredients such as collagen is improved, the combined application and synergistic effect of salidroside, collagen and EGCG are realized, and the provided salidroside / collagen / EGCG composition has the advantages of high stability, good transdermal effect, significant anti-aging effect and the like.

[0006] The object of the present application will be further illustrated by the following detailed description.

[0007] The present application provides a salidroside composition with a core-shell-shell structure, and a preparation method thereof, which comprises the following steps:

[0008] 1) EGCG inclusion: β-cyclodextrin and EGCG are added to water in a mass ratio of (1.15-1.25):1, and ultrasonic treatment is performed to form a β-cyclodextrin-EGCG inclusion complex;

[0009] 2) Liposome assembly: soybean phospholipid and cholesterol are weighed and dissolved in chloroform to form a uniform solution; salidroside is weighed and dissolved in the uniform solution, and under vacuum and heating conditions, chloroform is removed by rotary evaporation to form a soybean phospholipid-cholesterol film on the container wall. Then, the β-cyclodextrin-EGCG inclusion complex and PBS buffer are added to the container, and hydration is performed to form liposomes wrapping the salidroside and EGCG inclusion complex;

[0010] 3) Shell loading: collagen is weighed and mixed uniformly with a chitosan solution to form a chitosan-collagen complex; the complex is added dropwise to the liposomes, and the chitosan-collagen complex is coated on the outer layer of the liposomes by electrostatic adsorption to form a core-shell-shell three-layer structure; centrifugal purification is performed to obtain a salidroside composition with a core-shell-shell structure.

[0011] Preferably, the mass ratio is (1.18-1.22):1. More preferably, the mass ratio is 1.2:1.

[0012] Preferably, the frequency of the ultrasonic treatment is 35-45 kHz, and the time is 20-45 min. The effect of ultrasonic treatment is to promote the interaction between β-cyclodextrin and EGCG molecules by the energy generated by high-frequency vibration, to accelerate the speed of EGCG into the cavity of β-cyclodextrin, and thus to improve the inclusion efficiency. More preferably, the frequency of the ultrasonic treatment is 40 kHz, and the time is 30 min; it has been detected that under this condition, the inclusion rate of EGCG is as high as 92% or more, and a stable β-cyclodextrin-EGCG inclusion compound is formed.

[0013] Preferably, the mass ratio of the soybean phospholipid to the cholesterol is (65-75):(25-35). More preferably, the mass ratio of the soybean phospholipid to the cholesterol is 70:30, and this ratio can form a liposome structure capable of maintaining the lamellar embedding of salidroside.

[0014] Preferably, the vacuum and heating conditions are: vacuum degree -0.1 to -0.06 MPa, 35-45℃. More preferably, the vacuum and heating conditions are: vacuum degree -0.08 MPa, 40℃.

[0015] Preferably, the temperature of the hydration is 43-47℃. More preferably, the temperature of the hydration is 45℃. A temperature around 45℃ is helpful for the formation and stability of the liposome, so that the salidroside can be smoothly embedded in the lamellar structure of the liposome, and the EGCG inclusion compound is distributed in the hydrophilic layer of the liposome.

[0016] Preferably, the molecular weight of the collagen is 65-67 kDa, the mass percentage concentration of the chitosan solution is 0.4%-0.6%, and the dosage ratio of the collagen to the chitosan solution is 45-55 mg:8-12 mL. During the mixing process, the collagen and the chitosan are combined to form a complex through electrostatic interaction. More preferably, the degree of deacetylation of the chitosan is ≥85%.

[0017] Preferably, the centrifugal purification comprises: centrifugation at 3500-4500 rpm for 10-20 min, and discarding the supernatant and washing the precipitate with PBS buffer pH 7.4. More preferably, the centrifugal purification comprises: centrifugation at 4000 rpm for 15 min.

[0018] Correspondingly, the application also provides a preparation method of the salidroside composition with the core-shell-shell structure, comprising the following steps:

[0019] 1) EGCG inclusion: β-cyclodextrin and EGCG are added to water in a mass ratio of (1.15-1.25):1, and ultrasonic treatment is performed to form a β-cyclodextrin-EGCG inclusion compound;

[0020] 2) Liposome assembly: soybean phospholipid and cholesterol are weighed and dissolved in chloroform to form a uniform solution; salidroside is weighed and dissolved in the uniform solution; under vacuum and heating, chloroform is removed by rotary evaporation to form a soybean phospholipid-cholesterol film on the wall of the container; then the β-cyclodextrin-EGCG inclusion compound and PBS buffer are added to the container, and hydration is performed to form liposomes encapsulating the salidroside and EGCG inclusion compound;

[0021] 3) Shell loading: collagen is weighed and mixed uniformly with the chitosan solution to form a chitosan-collagen complex; the complex is added dropwise to the liposomes, and the chitosan-collagen complex is coated on the outer layer of the liposomes by electrostatic adsorption to form a core-shell-shell three-layer structure; centrifugal purification is performed to obtain a salidroside composition with a core-shell-shell structure.

[0022] Further, the present application also provides the use of the salidroside composition with a core-shell-shell structure in the preparation of anti-aging / antioxidant cosmetics.

[0023] In addition, the present application also provides a cosmetic comprising the salidroside composition with a core-shell-shell structure.

[0024] Compared with the prior art, the beneficial effects of the present application include:

[0025] (1) The present application provides a rhodiolide composition with a core-shell-shell structure, by designing a core-shell-shell three-layer carrier structure, using beta-cyclodextrin, liposomes and chitosan-collagen complex to perform inclusion, assembly and loading in turn, first forming a stable beta-cyclodextrin-EGCG inclusion compound, then forming liposomes wrapping rhodiolide and EGCG inclusion compound, and then coating chitosan-collagen complex on the outer layer of the liposomes to form a core-shell-shell three-layer structure, solving the problems of charge conflict between EGCG and collagen and phase separation between rhodiolide and collagen, avoiding multi-component repulsion, and realizing the combined application and synergistic effect of rhodiolide, collagen and EGCG. The inner core uses beta-cyclodextrin to include EGCG, which uses the 0.65-0.78 nm cavity of beta-cyclodextrin to match the 0.7 nm molecular size of EGCG, completely wrapping EGCG, effectively isolating the negative charge of EGCG from the positive charge of collagen at pH<5, avoiding the combination and precipitation of the two, and at the same time, effectively protecting EGCG from light degradation. The middle layer of the liposome wraps rhodiolide, and the EGCG inclusion compound is distributed in the hydrophilic layer of the liposome. In particular, the liposome structure formed by soybean phospholipid and cholesterol at a mass ratio of 70:30 can maintain the layered embedding of rhodiolide, and the phase separation problem caused by the liposolubility of rhodiolide and the water solubility of collagen is effectively solved. The liposome acts as a middle barrier, making it difficult for rhodiolide and collagen to directly contact each other. Centrifugation experiments show that the precipitation rate of the system after using the present structure is significantly reduced compared with the precipitation rate of more than 60% of the system in the prior art, ensuring the stability of the system. The chitosan-collagen complex of the outer shell forms a positive charge shell with a zeta potential of +30 mV or more by using chitosan with a degree of deacetylation of 85% or more. The shell not only provides additional protection for the internal structure, but also interacts with the negative charge on the surface of the skin during use. The collagen in the prior art has a transdermal rate of less than 5% due to its large molecular weight, while in the present structure, the positive charge layer of chitosan can be closely connected with the skin, promoting transdermal penetration by electroosmosis, and creating favorable conditions for the penetration and delivery of active ingredients.

[0026] (2) The rhodiolide / collagen / EGCG composition provided by the present application has the advantages of high stability, good transdermal effect and significant anti-aging effect. In terms of anti-aging effect-related indicators, the experimental group using an essence substrate containing 5% (mass percentage) of the composition of the present application has a wrinkle volume reduction rate of 37.2% and a skin elasticity improvement of 43.1% after 28 days, while the control group only has a wrinkle volume reduction rate of 4.1% and a skin elasticity improvement of 7.3%. These data fully prove that the technical solution of the present application realizes the ternary synergistic anti-aging effect of rhodiolide, collagen and EGCG.

[0027] (3) The preparation method of the salidroside composition with the core-shell-shell structure is simple, easy to control, and realizes stable preparation of the salidroside composition with the core-shell-shell structure.

[0028] (4) In addition, the application also provides a use of the salidroside composition with the core-shell-shell structure in preparation of anti-aging / antioxidant cosmetics, and the anti-aging effect is far higher than that of the prior art simple mixing method, thereby providing a more efficient solution for anti-aging of cosmetics. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Transmission electron microscope detection result of the salidroside / collagen / EGCG composition with the core-shell-shell structure.

[0030] Figure 2 Particle size distribution detection result of the salidroside / collagen / EGCG composition with the core-shell-shell structure.

[0031] Figure 3 Transdermal absorption capacity test result of the salidroside / collagen / EGCG composition with the core-shell-shell structure. DETAILED DESCRIPTION

[0032] The application will be further described in detail below in combination with the drawings and examples.

[0033] In the application, the components, reagents and equipment involved are all conventional commercially available products, or can be obtained through conventional technical means in the art. For example: beta-cyclodextrin (analytical pure, Sigma-Aldrich Company), EGCG (purity ≥98%, Chengdu Manster Biological Technology Co., Ltd.), soybean phospholipid (food grade, Shanghai Yuanye Biological Technology Co., Ltd.), collagen (molecular weight 66 KDa, Peptide Source (Guangzhou) Biological Technology Co., Ltd.), chitosan (degree of deacetylation ≥85%, Qingdao Bozhihuilibo Biological Technology Co., Ltd.), DMEM culture medium (Gibco Company), HP-beta-cyclodextrin (analytical pure, Sigma-Aldrich Company), gelatin (food grade, Shanghai Maikelin Biological Technology Co., Ltd.).

[0034] Example 1: Salidroside composition with core-shell-shell structure and characterization thereof

[0035] The salidroside composition with the core-shell-shell structure has the following preparation method, which comprises the following steps:

[0036] 1) EGCG inclusion: take β-cyclodextrin 36 mg, EGCG 30 mg, place in a conical flask, add 10 mL deionized water, put the conical flask into an ultrasonic cleaner (power 300 W, frequency 40 kHz), ultrasonic treatment for 30 min, to form β-cyclodextrin-EGCG inclusion complex. During ultrasonic treatment, the cavities of β-cyclodextrin and EGCG molecules are combined by intermolecular forces, so that EGCG gradually enters the cavities of β-cyclodextrin to form stable β-cyclodextrin-EGCG inclusion complex; after ultrasonic treatment, the EGCG inclusion rate is detected by high performance liquid chromatography, which is 92.3%;

[0037] 2) Liposome assembly: take 70 mg of soybean phospholipid and 30 mg of cholesterol, dissolve in 20 mL of chloroform to form a uniform solution; take 10 mg of salidroside, dissolve in the uniform solution, and transfer to a flask in a rotary evaporator, rotate at 40°C and a vacuum degree of-0.08 MPa, and after the chloroform is completely volatilized, a uniform soybean phospholipid-cholesterol film is formed on the inner wall of the flask; add the β-cyclodextrin-EGCG inclusion complex prepared in step 1) and 10 mL of PBS buffer to the flask, and hydrate at 45°C water bath for 1 h, stir, and disperse the film again to form a liposome that encapsulates salidroside and EGCG inclusion complex;

[0038] 3) Shell loading: take 50 mg of collagen (molecular weight 66 KDa) and 10 mL of 0.5% mass percentage chitosan solution, and mix them on a magnetic stirrer at a stirring speed of 300 r / min for 30 min to form a complex; slowly drop the complex into the liposome solution prepared in step 2) that encapsulates salidroside and EGCG inclusion complex, and control the dropping rate to be 1 drop / s, and continue to stir for 1 h after the dropping is completed, so that the chitosan-collagen complex is evenly coated on the outer layer of the liposome by electrostatic adsorption to form a core-shell-shell three-layer structure. Finally, transfer the mixed solution to a centrifuge tube and centrifuge at a speed of 4000 rpm for 15 min, discard the supernatant, and wash the precipitate with PBS buffer for 3 times to obtain a salidroside / collagen / EGCG composition with a core-shell-shell structure. The salidroside / collagen / EGCG composition is detected by transmission electron microscopy, and the results are shown in Figure 1 Figure 2 ​As shown, the average particle size of the salidroside / collagen / EGCG composition is 118 nm, the polydispersity index PDI = 0.18, indicating that the particle size distribution is uniform and has good monodispersity, which is conducive to stable application in cosmetic systems.

[0039] Example 2 Anti-wrinkle efficacy test

[0040] Experimental materials and equipment: anti-aging essence containing 5% by mass of the composition of the present application (the salidroside / collagen / EGCG composition prepared in Example 1 is added to the essence base, and the specific formula is shown in Table 1), placebo (the same essence base without the composition of the present application), ANTERA 3D skin detector (Miravex Company, USA).

[0041] Table 1 Formula of anti-aging essence containing 5% by mass of the composition of the present application

[0042]

[0043] The preparation method of the anti-aging essence containing 5% by mass of the composition of the present application includes the following steps: 1) heat phase A to 82-85°C and stir to dissolve until transparent; 2) keep the main pot temperature at 82-85°C and stir for 30 min; 3) start the cooling water; 4) when the temperature of the main pot drops to 50°C, add phase B and stir evenly (note: phase B should be completely dissolved in a water bath at 53-58°C until transparent before use); 5) reduce the temperature of the main pot to 40°C, add the composition of the present application, and stir until uniform.

[0044] Scheme: 30 healthy women aged 35-50 years old with obvious wrinkles on the face were selected as experimental subjects through public recruitment. Before the experiment, all subjects signed an informed consent form and stopped using other anti-wrinkle cosmetics for 1 week. Using the half-face coating method, the subjects were randomly assigned to the left or right side of the face, and one side of the face was evenly coated with the anti-aging essence of the experimental group, and the other side of the face was coated with the placebo of the control group, 0.5 g each time, twice a day, for 28 days.

[0045] Data collection and analysis: Before the experiment and after 28 days of the experiment, the ANTERA 3D skin detector was used to scan and detect the same area on both sides of the face of the subjects under the same environmental conditions (temperature 25±1°C, humidity 50±5%). When detecting, the instrument automatically collects three-dimensional image data of the skin surface, and calculates the wrinkle volume and skin elasticity parameters through professional analysis software. The data were statistically analyzed by paired sample t test, and the results are shown in Table 2.

[0046] Table 2 Anti-wrinkle efficacy test results

[0047]

[0048] The experimental results show that the anti-aging essence containing the carrier of the application has a significant effect on reducing wrinkle volume and improving skin elasticity, and the difference compared with the placebo group has statistical significance (P < 0.05).

[0049] Example Three Anti-blue light synergy experiment

[0050] Experimental materials and equipment: human keratinocytes (HaCaT, Chinese Academy of Sciences Typical Culture Preservation Committee Cell Library), DMEM culture medium containing 0.1% mass percentage of the composition of the application (add the composition of the application to the DMEM culture medium, referred to as the third-embedded group), physical mixing group culture medium (add the physical mixture of equal concentrations of rhodioside, collagen and EGCG to the DMEM culture medium), blank group culture medium (DMEM culture medium), blue light irradiation instrument (wavelength 450nm, adjustable irradiation intensity, Nanjing Xujia Machinery Plant), ROS detection kit (Bi Yun Tian Biological Technology Co., Ltd.), IL-6 ELISA detection kit (R&D Systems Company, USA), enzyme label instrument (BioTek Company, USA), CO2 incubator (Thermo Fisher Scientific Company, USA).

[0051] Method: HaCaT cells in the logarithmic growth phase were inoculated in a 96-well plate at a density of 5×10 4 cells / well, and placed in a 37℃, 5% CO2 incubator for 24h. After the cells grew well, the experimental group (DMEM culture medium containing 0.1% mass percentage of the composition of the application), the control group (physical mixing group culture medium, blank group culture medium) were replaced, and 6 replicate wells were set in each group. After 24h of continuous culture, the 96-well plate was moved to the blue light irradiation instrument, and blue light irradiation (450nm, 30J / cm²) was performed. After irradiation, the cell culture supernatant was collected for detection of IL-6 inflammatory factors; at the same time, according to the ROS detection kit instructions, the intracellular ROS level was detected; according to the live and dead staining kit instructions, the survival of HaCaT cells was detected, and the results are shown in Table 3.

[0052] Table 3 Anti-blue light experiment results

[0053]

[0054] The experimental results show that the composition has strong metal ion chelating capacity, can chelate iron ions in cells under blue light irradiation, inhibit the occurrence of photo-Fenton reaction, thereby reduce the generation of ROS, and the effect is obviously better than that of the physical mixing group and the blank group; can also activate the Nrf2 pathway in cells, up-regulate the expression of antioxidant enzymes, and further enhance the antioxidant capacity of cells, the synergistic effect of the two can effectively reduce the damage of blue light to cells, improve the cell survival rate and inhibit the release of inflammatory factor IL-6, and the effect is obviously better than that of the physical mixing group and the blank group.

[0055] Example Four Stability Test

[0056] Experimental materials and methods: the following three groups of samples were prepared: β-cyclodextrin + liposome (without chitosan) group, the preparation method was similar to example one, but step 3) was not carried out; liposome + chitosan (without β-cyclodextrin) group, that is, step 1) was not carried out, and the salidroside was directly wrapped in the liposome and then the outer shell was loaded; the salidroside / collagen / EGCG composition (tertiary structure system) prepared in example one. The three groups of samples were respectively placed in a 45℃ constant temperature box for storage for 30 days, during which the appearance change of the samples was observed regularly, and after the storage was completed, the following detection was carried out: the precipitation rate was detected by centrifugal experiment (4000rpm, 15min); the sample was exposed to blue light (450nm, 30J / cm²) for 1h, and the color change was observed; the retention rate of active ingredients was determined by HPLC method, and the results are shown in table 4.

[0057] Table 4 Stability test results

[0058]

[0059] The experimental results show that: when the outer shell loading treatment is missing chitosan, the EGCG is easily oxidized and discolored due to the lack of protection of the positive charge antioxidant layer, and the activity retention rate is significantly reduced; when the inner layer EGCG inclusion treatment is missing, the salidroside stability decreases due to the lack of hydrophilic modification, and the precipitation phenomenon occurs; and the tertiary structure system of the application can effectively protect each active ingredient and maintain the stability of the system, which proves the superiority of the tertiary structure.

[0060] Example Five Transdermal Performance Test

[0061] FITC (isothiocyanate) preparation:

[0062] (1) Preparation of crosslinking reaction solution: 0.756g NaHCO3, 0.106g Na2CO3, 0.736g NaCl, ultrapure water to 100mL, pH 8.7;

[0063] (2) Cross-linking reaction termination solution: 5 M NH4Cl aqueous solution;

[0064] (3) FITC solution: a certain amount of FITC was dissolved in DMSO to prepare a solution with a concentration of 10 mg / mL, which was prepared and used immediately;

[0065] Preparation of test samples and control samples:

[0066] (1) 30 mg of each of the test collagen samples was reconstituted with 15 mL of the cross-linking reaction solution, 500 μL of the FITC solution was added, and the mixture was mixed and incubated at 4°C in the dark overnight;

[0067] (2) 200 μL of the cross-linking reaction termination solution (final concentration 50 mM) was added, and the mixture was incubated at 4°C in the dark for 3 h;

[0068] (3) The mixture was dialyzed for 12 h, and the dialysis medium was replaced every 2 h to remove unbound FITC;

[0069] (4) After dialysis, the FITC-labeled test collagen solution was collected;

[0070] (5) The FITC-labeled collagen was used to prepare triple-wrapped liposomes and physical mixtures, respectively.

[0071] Preparation of the FITC control sample (without collagen solution): 5 mg of FITC was dissolved in 500 μL of DMSO, and then diluted with PBS buffer to 20 mL, with a final concentration of 0.25 mg / mL, and prepared in the dark.

[0072] In vitro transdermal semi-quantitative analysis--Franz diffusion cell method:

[0073] (1) Receiving solution: PBS buffer, 15 mL of receiving solution was added to the receiving cell;

[0074] (2) Diffusion cell: 2 mL of each of the FITC solution, FITC-labeled collagen triple-embedded and physical mixture was added to the diffusion cell as the sample to be determined, and the diffusion cell was covered with a sealing film to prevent evaporation of the solution;

[0075] (3) The stratum corneum of the pig skin was upward, and the lower skin was in contact with the receiving cell liquid, and the receiving cell was in contact with the skin to avoid air bubbles;

[0076] (4) The water bath temperature of the diffusion instrument was 37°C, and the stirring speed was 350 rpm / min;

[0077] (5) The sample in the receiving cell was collected every 4 h, 0.5 mL, and the same volume of receiving solution was supplemented, for a total of 24 h;

[0078] (6) The experiment should be performed in the dark to prevent quenching of the FITC fluorescence.

[0079] Determination of FITC fluorescence intensity:

[0080] (1) 100 μL of the receiving solution was taken from the sampling of the receiving pool of each group into one well of a 96-well plate, with three parallel wells for each group;

[0081] (2) The fluorescence intensity of the sample in the well was detected using a bioluminescence instrument in the dark.

[0082] The test results are shown in Table 2, and the composition of the present application can promote the transdermal absorption of collagen through three-stage embedding. Figure 3

[0083] Performance comparison with alternative materials

[0084] Experimental materials and methods: The following three groups of samples were prepared respectively: the group of the present application (the rhodiolosyl / collagen / EGCG composition prepared in Example 1); the HP-β-cyclodextrin replacement group, in which the β-cyclodextrin in Example 1 was replaced with HP-β-cyclodextrin, and the rest of the preparation steps were the same as in Example 1; the gelatin instead of chitosan group, in which chitosan was replaced with gelatin, and the rest of the preparation steps were the same as in Example 1. The following performance tests were performed on the three groups of samples respectively: the encapsulation efficiency was determined by HPLC method; the transdermal rate was determined by Franz diffusion cell (ex vivo pig skin) experiment; the samples were centrifuged at 4000 rpm for 15 min, and the centrifugal stability was observed; the Zeta potential at pH 6.5 was determined using a Zeta potential instrument (Malvern Zetasizer Nano ZS, Malvern Panalytical, UK), and the results are shown in Table 5.

[0085] Table 5 Comparison results of performance with alternative materials

[0086]

[0087] The experimental results show that the pore size of HP-β-cyclodextrin is too large (1.8 nm), which cannot tightly encapsulate EGCG, resulting in easy leakage of EGCG in the system, thereby reducing the encapsulation efficiency and transdermal rate; the isoelectric point of gelatin is pH 4.7-5.2, and the positive charge is insufficient at pH 6.5, which cannot effectively form a stable positive charge shell, affecting the transdermal performance and stability of the carrier, and the gelation phenomenon also indicates that gelatin cannot play an ideal protective and promoting role in the system.

[0088] Comparison of efficacy with commercially available competitors

[0089] ​Experimental materials and methods: sample A is an essence base added with 5% by mass content of Schizosaccharomyces pombe fermentation product (the difference from sample C is that the Schizosaccharomyces pombe fermentation product is added, and the composition of the application is not contained), sample B is an essence base containing 15% by mass content of VC and 1% by mass content of VE, and sample C is an anti-aging essence containing 5% by mass content of the composition of the application. 60 healthy women aged 35-50 years old with similar skin conditions were selected as experimental subjects by public recruitment and were randomly divided into 3 groups, 20 people in each group. Before the experiment, all the subjects signed the informed consent form and stopped using other anti-wrinkle, whitening and moisturizing cosmetics for 1 week. Each subject in each group used the corresponding sample once a day in the morning and evening, and the amount used each time was 0.5g, which lasted for 28 days. Before the experiment started and after the experiment ended for 28 days, the wrinkle depth, the number and area of ultraviolet spots, the amount of transepidermal water loss and other parameters of the subjects were detected. The data were statistically processed by one-way analysis of variance. The results of the 28-day human body test are shown in Table 6.

[0090] Table 6 Comparison results of efficacy with commercially available competitors

[0091]

[0092] The experimental results show that compared with commercially available well-known competitors, the rhodiolin / collagen / EGCG composition provided by the application has more significant effects in reducing wrinkle depth, reducing ultraviolet spots and reducing transepidermal water loss, and the difference has statistical significance (P < 0.05 or P < 0.01).

[0093] The above is a further detailed description of the application in combination with specific preferred embodiments, and the specific implementation of the application cannot be limited to these descriptions. For ordinary skilled persons in the technical field to which the application belongs, without departing from the concept of the application, a number of simple deductions or substitutions can be made, which should be regarded as falling within the protection scope of the application.

Claims

1. A salidroside composition having a core-shell-shell structure, characterized in that: The preparation method comprises the following steps: 1) EGCG inclusion: β-cyclodextrin and EGCG are added into appropriate water in a mass ratio of (1.15-1.25):1, and ultrasonic treatment is performed to form a β-cyclodextrin-EGCG inclusion compound; 2) liposome assembly: soybean phospholipid and cholesterol are weighed and dissolved in chloroform to form a uniform solution; salidroside is weighed and dissolved in the uniform solution, and under vacuum and heating conditions, chloroform is removed by rotary evaporation to form a soybean phospholipid-cholesterol film on the wall of the container, then the β-cyclodextrin-EGCG inclusion compound and PBS buffer are added into the container, and hydration is performed to form a liposome wrapping salidroside and the EGCG inclusion compound; 3) shell loading: collagen is weighed and uniformly mixed with a chitosan solution to form a chitosan-collagen compound; the compound is added dropwise into the liposome, and the chitosan-collagen compound is coated on the outer layer of the liposome by electrostatic adsorption to form a core-shell-shell three-layer structure; centrifugal purification is performed to obtain a salidroside composition with a core-shell-shell structure; The mass ratio of the soybean phospholipid to the cholesterol is (65-75):(25-35). The molecular weight of the collagen is 65-67 kDa, the mass percentage concentration of the chitosan solution is 0.4%-0.6%, the dosage ratio of the collagen to the chitosan solution is 45-55 mg:8-12 mL, and the degree of deacetylation of the chitosan is ≥85%.

2. The ginsenoside composition having a core-shell-shell structure according to claim 1, characterized by: The mass ratio in the step 1) is (1.18-1.22):

1.

3. The ginsenoside composition having a core-shell-shell structure according to claim 1, characterized by: The ultrasonic treatment is performed at a frequency of 35-45 kHz for 20-45 min.

4. The ginsenoside composition having a core-shell-shell structure according to any one of claims 1 to 3, characterized in that: The vacuum and heating conditions are: vacuum degree -0.1 to -0.06 MPa, 35-45℃.

5. The ginsenoside composition having a core-shell-shell structure according to any one of claims 1 to 3, characterized in that: The hydration temperature is 43-47℃.

6. The method of preparing a ginsenoside composition having a core-shell-shell structure according to claim 1, characterized by: The preparation method comprises the following steps: 1) EGCG inclusion: β-cyclodextrin and EGCG are added into appropriate water in a mass ratio of (1.15-1.25):1, and ultrasonic treatment is performed to form a β-cyclodextrin-EGCG inclusion compound; 2) liposome assembly: soybean phospholipid and cholesterol are weighed and dissolved in chloroform to form a uniform solution; salidroside is weighed and dissolved in the uniform solution, and under vacuum and heating conditions, chloroform is removed by rotary evaporation to form a soybean phospholipid-cholesterol film on the wall of the container, then the β-cyclodextrin-EGCG inclusion compound and PBS buffer are added into the container, and hydration is performed to form a liposome wrapping salidroside and the EGCG inclusion compound; 3) shell loading: collagen is weighed and uniformly mixed with a chitosan solution to form a chitosan-collagen compound; the compound is added dropwise into the liposome, and the chitosan-collagen compound is coated on the outer layer of the liposome by electrostatic adsorption to form a core-shell-shell three-layer structure; centrifugal purification is performed to obtain a salidroside composition with a core-shell-shell structure.

7. Use of the salidroside composition with a core-shell-shell structure according to claim 1 in the preparation of anti-aging cosmetics.

8. A cosmetic product, characterized by: The salidroside composition with a core-shell-shell structure according to any one of claims 1 to 5. The salidroside composition with a core-shell-shell structure according to any one of claims 1 to 5.

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