Salidroside composition with core-shell-shell structure as well as preparation method and application of salidroside composition

Abstract

The invention provides a salidroside composition with a core-shell-shell structure, and a preparation method of the salidroside composition comprises the following steps: 1) EGCG (epigallocatechin gallate) clathration: adding beta-cyclodextrin and EGCG into water, and carrying out ultrasonic treatment to form a clathrate compound; 2) liposome assembly: weighing soybean phospholipid and cholesterol, and dissolving in chloroform to form a uniform solution; the preparation method comprises the following steps: weighing salidroside, dissolving the salidroside in a uniform solution, carrying out rotary evaporation to remove chloroform, then adding an inclusion compound and a PBS (Phosphate Buffer Solution) into a container, and hydrating to form lipidosome wrapping the salidroside and the inclusion compound; 3) shell loading: weighing collagen, and uniformly mixing the collagen with the chitosan solution to form a compound; the salidroside composition with the core-shell-shell structure is obtained by dropwise adding the compound into the lipidosome, coating the outer layer of the lipidosome with the compound and carrying out centrifugal purification. The invention belongs to the technical field of cosmetic raw materials, and provides a salidroside composition which has the advantages of high stability, good transdermal effect, remarkable anti-aging effect and the like.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic raw material technology, and particularly relates to a rhodioloside composition with a core-shell-shell structure, its preparation method and uses. Background Technology

[0002] In the field of cosmetic raw materials, rhodioloside, collagen, and epigallocatechin gallate (EGCG) have attracted much attention due to their respective excellent anti-aging effects and play an important role in cosmetics and other fields. Rhodioloside has antioxidant and cell metabolism-regulating effects; collagen can maintain skin elasticity and firmness; EGCG is a highly effective antioxidant that can scavenge free radicals and inhibit inflammatory responses. However, rhodioloside has poor long-term stability in aqueous solutions and is easily affected by factors such as light and oxidation. Recombinant collagen typically has a molecular weight of 30-500 kDa. According to Franz diffusion cell data, its transdermal penetration rate is less than 5%. Large collagen molecules have difficulty penetrating the stratum corneum barrier and cannot effectively penetrate the deep layers of the skin, resulting in a significant reduction in anti-aging effects. EGCG is sensitive to light, with a half-life of less than 24 hours. High-performance liquid chromatography (HPLC) detection shows that its degradation rate is as high as 80% after light exposure, leading to a large loss of active ingredients and making it difficult to fully exert its anti-aging effects.

[0003] To improve the stability of rhodioloside, existing technologies mainly prepare it into liposomes. Chinese patent application CN 113041169 A discloses a rhodioloside liposome lyophilized powder, prepared from rhodioloside, egg yolk lecithin, and cholesterol, which exhibits good stability. Since EGCG carries a negative charge, while collagen carries a positive charge at pH < 5, directly mixing EGCG and collagen will cause electrostatic attraction and precipitation. To overcome the problems existing in the application of collagen and EGCG, Chinese patent application CN 117137818 A discloses a self-assembled microsphere of EGCG and recombinant collagen. This technology achieves the combined use of EGCG and recombinant collagen through self-assembly. Specifically, EGCG and the proline and hydroxyproline of recombinant collagen (COL) are bound by intermolecular hydrogen bonds and π-bonds (non-covalent bonds) to self-assemble into nanospheres. This improves collagen permeability while inhibiting EGCG oxidation and reducing EGCG cytotoxicity. Because rhodioloside is fat-soluble, it presents a compatibility issue with water-soluble collagen. Mixing them can easily lead to phase separation, affecting the stability and homogeneity of the system and resulting in unstable product quality. Therefore, it is difficult to achieve combined applications of rhodioloside, collagen, and EGCG through simple mixing.

[0004] Therefore, it is of great significance to provide a rhodioloside composition with a core-shell-shell structure, its preparation method and uses. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a rhodioloside composition with a core-shell-shell structure (hereinafter referred to as the composition of this invention). By designing a core-shell-shell three-layer carrier structure, β-cyclodextrin, liposomes, and chitosan-collagen complex are used sequentially for inclusion, assembly, and loading, solving the charge conflict between EGCG and collagen and the phase separation problem between rhodioloside and collagen. At the same time, it effectively protects EGCG from photodegradation, improves the transdermal permeability of active ingredients such as collagen, and realizes the combined application and synergistic effect of rhodioloside, collagen, and EGCG. The provided rhodioloside / collagen / EGCG composition has the advantages of high stability, good transdermal effect, and significant anti-aging effect.

[0006] The objectives of this invention will be further explained by the following detailed description.

[0007] This invention provides a rhodioloside composition having a core-shell-shell structure, the preparation method of which includes the following steps: 1) EGCG inclusion complex: β-cyclodextrin and EGCG were added to an appropriate amount of water at a mass ratio of (1.15-1.25):1 and ultrasonically treated to form a β-cyclodextrin-EGCG inclusion complex; 2) Liposome assembly: Weigh soybean lecithin and cholesterol, dissolve them in chloroform to form a homogeneous solution; weigh rhodioloside, dissolve it in the homogeneous solution, remove chloroform by rotary evaporation under vacuum and heating conditions, forming a soybean lecithin-cholesterol film on the container wall, then add the β-cyclodextrin-EGCG inclusion complex and PBS buffer to the container, hydrate, and form liposomes encapsulating rhodioloside and EGCG inclusion complex; 3) Shell loading: Weigh collagen and mix it evenly with chitosan solution to form a chitosan-collagen complex; add the complex dropwise to the liposomes, and through electrostatic adsorption, the chitosan-collagen complex coats the outer layer of the liposomes to form a core-shell-shell three-layer structure; centrifuge and purify to obtain a rhodioloside composition with a core-shell-shell structure.

[0008] Preferably, the mass ratio is (1.18-1.22):1. More preferably, the mass ratio is 1.2:1.

[0009] Preferably, the ultrasonic treatment frequency is 35-45 kHz and the time is 20-45 min. The effect of ultrasound is to promote the interaction between β-cyclodextrin and EGCG molecules through the energy generated by high-frequency vibration, thereby accelerating the entry of EGCG into the β-cyclodextrin cavity and improving the inclusion efficiency. More preferably, the ultrasonic treatment frequency is 40 kHz and the time is 30 min; after testing, the inclusion rate of EGCG under these conditions is as high as 92% or more, forming a stable β-cyclodextrin-EGCG inclusion complex.

[0010] Preferably, the mass ratio of soybean lecithin to cholesterol is (65-75):(25-35). More preferably, the mass ratio of soybean lecithin to cholesterol is 70:30, and the liposome structure formed by this ratio can maintain the layered embedding of rhodioloside.

[0011] Preferably, the vacuum and heating conditions are: vacuum degree -0.1 to -0.06 MPa, 35-45°C. More preferably, the vacuum and heating conditions are: vacuum degree -0.08 MPa, 40°C.

[0012] Preferably, the hydration temperature is 43-47°C. More preferably, the hydration temperature is 45°C. A temperature around 45°C facilitates the formation and stabilization of liposomes, allowing rhodioloside to be readily embedded in the layered structure of the liposomes, with the EGCG inclusion complex distributed in the hydrophilic layer of the liposomes.

[0013] Preferably, the collagen has a molecular weight of 65-67 kDa, and the chitosan solution has a mass percentage concentration of 0.4%-0.6%; the ratio of collagen to chitosan solution is 45-55 mg: 8-12 mL. During mixing, the collagen and chitosan bind together through electrostatic interactions to form a complex. More preferably, the degree of deacetylation of the chitosan is ≥85%.

[0014] Preferably, the centrifugal purification includes: centrifuging at 3500-4500 rpm for 10-20 min, discarding the supernatant, and washing the precipitate with PBS buffer at pH 7.4. More preferably, the centrifugal purification includes: centrifuging at 4000 rpm for 15 min.

[0015] Accordingly, the present invention also provides a method for preparing the rhodioloside composition having a core-shell-shell structure, comprising the following steps: 1) EGCG inclusion complex: β-cyclodextrin and EGCG were added to an appropriate amount of water at a mass ratio of (1.15-1.25):1 and ultrasonically treated to form a β-cyclodextrin-EGCG inclusion complex; 2) Liposome assembly: Weigh soybean lecithin and cholesterol, dissolve them in chloroform to form a homogeneous solution; weigh rhodioloside, dissolve it in the homogeneous solution, remove chloroform by rotary evaporation under vacuum and heating conditions, forming a soybean lecithin-cholesterol film on the container wall, then add the β-cyclodextrin-EGCG inclusion complex and PBS buffer to the container, hydrate, and form liposomes encapsulating rhodioloside and EGCG inclusion complex; 3) Shell loading: Weigh collagen and mix it evenly with chitosan solution to form a chitosan-collagen complex; add the complex dropwise to the liposomes, and through electrostatic adsorption, the chitosan-collagen complex coats the outer layer of the liposomes to form a core-shell-shell three-layer structure; centrifuge and purify to obtain a rhodioloside composition with a core-shell-shell structure.

[0016] Furthermore, the present invention also provides the use of the rhodioloside composition having a core-shell-shell structure in the preparation of anti-aging / antioxidant cosmetics.

[0017] In addition, the present invention also provides a cosmetic comprising the rhodioloside composition having a core-shell-shell structure.

[0018] Compared with the prior art, the beneficial effects of the present invention include: (1) This invention provides a rhodioloside composition with a core-shell-shell structure. By designing a core-shell-shell three-layer carrier structure, β-cyclodextrin, liposomes and chitosan-collagen complex are used to encapsulate, assemble and load the components in sequence. First, a stable β-cyclodextrin-EGCG inclusion complex is formed. Then, liposomes are formed to encapsulate the rhodioloside and EGCG inclusion complex. Finally, the chitosan-collagen complex is coated on the outer layer of the liposomes to form a core-shell-shell three-layer structure. This solves the problems of charge conflict between EGCG and collagen and phase separation between rhodioloside and collagen, avoids mutual repulsion of multiple components, and realizes the combined application and synergistic effect of rhodioloside, collagen and EGCG. The core utilizes β-cyclodextrin to encapsulate EGCG. The 0.65-0.78 nm cavity of β-cyclodextrin matches the 0.7 nm molecular size of EGCG, completely encapsulating it. This effectively isolates the negative charge of EGCG from the positive charge of collagen at pH < 5, preventing their binding and precipitation. Simultaneously, it effectively protects EGCG from photodegradation. The intermediate liposome layer encapsulates rhodioloside, with the EGCG inclusion complex distributed within the hydrophilic layer of the liposome. Specifically, the liposome structure formed by a 70:30 mass ratio of soybean lecithin and cholesterol maintains the layered embedding of rhodioloside. The phase separation problem caused by the lipid solubility of rhodioloside and the water solubility of collagen is effectively solved with this structure. The liposome acts as an intermediate barrier, making direct contact between rhodioloside and collagen difficult. Centrifugation experiments show that compared to the precipitation rate of over 60% in existing systems, the precipitation rate of the system using this structure is significantly reduced, ensuring the stability of the system. The chitosan-collagen complex in the outer shell forms a positively charged shell with a zeta potential ≥ +30mV through chitosan with a degree of deacetylation ≥ 85%. This shell not only provides additional protection for the internal structure but also interacts with the negatively charged skin surface during use. Existing collagen technologies often have a transdermal permeability of less than 5% due to their large molecular weight. However, in this structure, the positively charged chitosan layer can tightly bind to the skin, promoting transdermal absorption through electroosmosis and creating favorable conditions for the penetration and delivery of active ingredients.

[0019] (2) The rhodioloside / collagen / EGCG composition provided by this invention has advantages such as high stability, good transdermal effect, and significant anti-aging effect. Regarding anti-aging efficacy indicators, the experimental group using an essence matrix containing 5% (by mass) of the composition of this invention showed a 37.2% reduction in wrinkle volume and a 43.1% increase in skin elasticity after 28 days, while the control group showed only 4.1% and 7.3%, respectively. These data fully demonstrate that the technical solution of this invention achieves a synergistic anti-aging effect of rhodioloside, collagen, and EGCG.

[0020] (3) The preparation method of the rhodioloside composition with core-shell-shell structure provided by the present invention is relatively simple and easy to control, and the stable preparation of the rhodioloside composition with core-shell-shell structure is realized.

[0021] (4) In addition, the present invention also provides the use of the rhodioloside composition with the core-shell-shell structure in the preparation of anti-aging / antioxidant cosmetics. The anti-aging effect is far superior to the simple mixing method in the prior art, providing a more efficient solution for anti-aging cosmetics. Attached Figure Description

[0022] Figure 1 The transmission electron microscopy results of the rhodioloside / collagen / EGCG composition with a core-shell-shell structure of the present invention.

[0023] Figure 2 The particle size distribution detection results of the rhodioloside / collagen / EGCG composition with a core-shell-shell structure of the present invention.

[0024] Figure 3 The transdermal absorption capacity test results of the rhodioloside / collagen / EGCG composition with a core-shell-shell structure of the present invention. Detailed Implementation

[0025] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments.

[0026] In this invention, all components, reagents, and equipment are commercially available products or can be obtained through conventional techniques in the field. Examples include: β-cyclodextrin (analytical grade, Sigma-Aldrich), EGCG (purity ≥98%, Chengdu Manster Biotechnology Co., Ltd.), soybean lecithin (food grade, Shanghai Yuanye Biotechnology Co., Ltd.), collagen (molecular weight 66 kDa, Peptide Source (Guangzhou) Biotechnology Co., Ltd.), chitosan (degree of deacetylation ≥85%, Qingdao Bozhi Huili Biotechnology Co., Ltd.), DMEM culture medium (Gibco), HP-β-cyclodextrin (analytical grade, Sigma-Aldrich), and gelatin (food grade, Shanghai Maclean Biochemical Technology Co., Ltd.).

[0027] Example 1: Rhodioloside composition with core-shell-shell structure and its characterization The preparation method of a rhodioloside composition having a core-shell-shell structure includes the following steps: 1) EGCG inclusion complex: Weigh 36 mg of β-cyclodextrin and 30 mg of EGCG into an Erlenmeyer flask, add 10 mL of deionized water, and place the flask in an ultrasonic cleaner (300 W, 40 kHz) for 30 min to form a β-cyclodextrin-EGCG inclusion complex. During the ultrasonic process, the β-cyclodextrin cavity and EGCG molecules interact through intermolecular forces, allowing EGCG to gradually enter the β-cyclodextrin cavity and form a stable β-cyclodextrin-EGCG inclusion complex. After ultrasonication, the EGCG inclusion rate was determined to be 92.3% by high-performance liquid chromatography. 2) Liposome assembly: Weigh 70 mg of soybean lecithin and 30 mg of cholesterol, dissolve them in 20 mL of chloroform to form a homogeneous solution; weigh 10 mg of rhodioloside, dissolve it in the homogeneous solution, transfer it to a globular flask of a rotary evaporator, and rotary evaporate it at 40 °C and a vacuum of -0.08 MPa. After the chloroform has completely evaporated, a uniform soybean lecithin-cholesterol film is formed on the inner wall of the globular flask; add the β-cyclodextrin-EGCG inclusion complex prepared in step 1) and 10 mL of PBS buffer to the flask, hydrate it in a 45 °C water bath for 1 h, stir to redisperse the film, and form liposomes encapsulating the rhodioloside and EGCG inclusion complex; 3) Shell Loading: Weigh 50 mg of collagen (molecular weight 66 kDa) and 10 mL of 0.5% (w / w) chitosan solution. Mix the two solutions on a magnetic stirrer at 300 rpm for 30 min to ensure they fully combine and form a complex. Slowly add this complex dropwise to the liposome solution containing the rhodioloside and EGCG complex prepared in step 2), with a drop rate controlled at 1 drop / s. After the addition is complete, continue stirring for 1 h to allow the chitosan-collagen complex to uniformly coat the outer layer of the liposomes through electrostatic adsorption, forming a core-shell-shell three-layer structure. Finally, transfer the mixture to a centrifuge tube and centrifuge at 4000 rpm for 15 min. Discard the supernatant and wash the precipitate three times with PBS buffer to obtain the core-shell-shell structured rhodioloside / collagen / EGCG composition. The rhodioloside / collagen / EGCG composition was analyzed using transmission electron microscopy, and the results are as follows: Figure 1 As shown, the rhodioloside / collagen / EGCG composition prepared in this invention clearly exhibits a three-layer structure distribution. Dynamic light scattering (DLS, Malvern Zetasizer Nano ZS, Malvern Panacol, UK) was used for detection, and the results are as follows... Figure 2 As shown, the average particle size of the rhodioloside / collagen / EGCG composition is 118 nm, and the polydispersity index (PDI) is 0.18, indicating that the particle size distribution is uniform and the composition has good monodispersity, which is beneficial for its stable application in cosmetic systems.

[0028] Example 2: Anti-wrinkle efficacy test Experimental materials and equipment: an anti-aging serum containing 5% by weight of the composition of the present invention (the rhodioloside / collagen / EGCG composition prepared in Example 1 was added to the serum matrix, the specific formula of which is shown in Table 1), a placebo (the same serum matrix without the composition of the present invention), and an ANTERA 3D skin analyzer (Miravex Corporation, USA).

[0029] Table 1. Anti-aging essence formulations containing 5% by weight of the composition of the present invention.

[0030] The preparation method of the anti-aging essence containing 5% by mass of the composition of the present invention includes the following steps: 1) Heat phase A to 82-85℃ and stir until dissolved and transparent; 2) Maintain the temperature of the main pot at 82-85℃ and stir for 30 minutes; 3) Turn on the cooling water; 4) When the temperature of the main pot drops to 50℃, add phase B and stir evenly (Note: phase B must be completely dissolved in a water bath at 53℃-58℃ until transparent before use); 5) When the temperature of the main pot drops to 40℃, add the composition of the present invention and stir until well mixed.

[0031] Protocol: Thirty healthy women aged 35-50 with noticeable facial wrinkles were selected through open recruitment as participants. Before the experiment, all participants signed informed consent forms and discontinued using other anti-wrinkle cosmetics for one week. A half-face application method was used, with participants randomly assigned to either the left or right side of their face. One side of the face was evenly coated with the anti-aging serum (experimental group), while the other side received a placebo (control group). Applications were made twice daily, morning and evening, with each application consisting of 0.5g, for 28 days.

[0032] Data Collection and Analysis: Before the experiment and after the 28-day experiment, the ANTERA 3D skin analyzer was used to scan the same area on both sides of the subjects' faces under the same environmental conditions (temperature 25±1℃, humidity 50±5%). During the scan, the instrument automatically acquired three-dimensional image data of the skin surface, and wrinkle volume and skin elasticity parameters were calculated using professional analysis software. Paired-samples t-tests were used to perform statistical analysis on the data, and the results are shown in Table 2.

[0033] Table 2 Anti-wrinkle efficacy test results

[0034] Experimental results show that the anti-aging essence containing the carrier of this invention has significant effects in reducing wrinkle volume and improving skin elasticity, and the difference is statistically significant compared with the placebo group (P < 0.05).

[0035] Example 3: Anti-blue light synergistic experiment

[0036] Experimental materials and equipment: human keratinocytes (HaCaT, Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee), DMEM medium containing 0.1% by mass of the composition of this invention (the composition of this invention was added to DMEM medium, referred to as the tertiary embedding group), physical mixing group medium (equal concentrations of rhodioloside, collagen and EGCG were directly physically mixed and then added to DMEM medium), blank group medium (DMEM medium), blue light irradiation instrument (wavelength 450nm, adjustable irradiation intensity, Nanjing Xujiang Electromechanical Plant), ROS detection kit (Beyotime Biotechnology Co., Ltd.), IL-6 ELISA detection kit (R&D Systems, USA), microplate reader (BioTek, USA), CO2 incubator (Thermo Fisher Scientific, USA).

[0037] Methods: HaCaT cells in logarithmic growth phase were injected with 5 × 10⁻⁶ cells. 4 Cells were seeded at a density of [number] cells / well in 96-well plates and cultured at 37°C with 5% CO2 for 24 hours. After good cell adhesion and growth, the medium was replaced with either the experimental group (DMEM medium containing 0.1% by weight of the composition of this invention) or the control group (physically mixed medium and blank medium), with 6 replicates per group. After another 24 hours of culture, the 96-well plates were transferred to a blue light irradiation instrument for blue light irradiation (450nm, 30J / cm²). After irradiation, the cell culture supernatant was collected for the detection of IL-6 inflammatory factor; simultaneously, the intracellular ROS level was detected according to the ROS detection kit instructions; and the viability of HaCaT cells was detected according to the live / dead staining kit instructions. The results are shown in Table 3.

[0038] Table 3 Results of blue light protection experiment

[0039] Experimental results show that the composition of this invention has a strong metal ion chelating ability. Under blue light irradiation, it can chelate iron ions in cells, inhibit the occurrence of photo-Fenton reaction, and thus reduce ROS production. The effect is significantly better than that of the physical mixture group and the blank group. It can also activate the Nrf2 pathway in cells, upregulate the expression of antioxidant enzymes, and further enhance the antioxidant capacity of cells. The two work synergistically to effectively reduce the damage caused by blue light to cells, improve cell survival rate, and inhibit the release of inflammatory factor IL-6. The effect is significantly better than that of the physical mixture group and the blank group.

[0040] Example 4: Stability Test

[0041] Experimental Materials and Methods: Three groups of samples were prepared: β-cyclodextrin + liposomes (chitosan-free) group, prepared similarly to Example 1, but without the shell loading treatment in step 3); liposomes + chitosan (β-cyclodextrin-free) group, i.e., without the EGCG inclusion treatment in step 1), rhodioloside was directly encapsulated in liposomes before shell loading; and the rhodioloside / collagen / EGCG composition (tertiary structure system) prepared in Example 1. The three groups of samples were stored in a 45℃ incubator for 30 days, during which the appearance of the samples was observed periodically. After storage, the following tests were performed: precipitation rate was detected by centrifugation (4000 rpm, 15 min); color change was observed after exposing the samples to blue light (450 nm, 30 J / cm²) for 1 h; and the retention rate of active ingredients was determined by HPLC. The results are shown in Table 4.

[0042] Table 4 Stability Test Results

[0043] Experimental results show that when the outer shell of chitosan is missing, EGCG is easily oxidized and discolored due to the lack of a positively charged antioxidant layer, and the activity retention rate is significantly reduced. When EGCG without the inner layer is included, rhodioloside has decreased stability in the system due to the lack of hydrophilic modification, resulting in precipitation. However, the tertiary structure system of this invention can effectively protect each active ingredient and maintain the stability of the system, demonstrating the superiority of the tertiary structure.

[0044] Example 5: Transdermal Performance Test

[0045] FITC (isothiocyanate) preparation: (1) Preparation of cross-linking reaction solution: 0.756g NaHCO3, 0.106g Na2CO3, 0.736g NaCl, ultrapure water to a final volume of 100 mL, pH 8.7; (2) Crosslinking reaction termination solution: 5 M NH4Cl aqueous solution; (3) FITC solution: Weigh a certain amount of FITC and dissolve it in DMSO to prepare a concentration of 10 mg / mL. Prepare and use immediately. Preparation of test and control samples: (1) Dissolve 30 mg of each collagen sample in 15 mL of cross-linking reaction solution, add 500 μL of FITC solution, mix well, and incubate overnight at 4°C in the dark. (2) Add 200 μL of crosslinking reaction termination solution (final concentration of 50 mM), and let stand at 4°C in the dark for 3 h; (3) Dialyze for 12 hours, changing the dialysis medium every 2 hours to remove unbound FITC; (4) Collect the FITC-labeled collagen solution submitted for testing after dialysis; (5) Using FITC-labeled collagen, triple-encapsulated liposomes and physical mixtures were prepared respectively.

[0046] Preparation of FITC control sample (collagen-free solution): Weigh 5 mg of FITC and dissolve it in 500 μL of DMSO, then adjust the volume to 20 mL with PBS buffer to a final concentration of 0.25 mg / mL. Prepare in the dark.

[0047] In vitro transdermal semi-quantitative analysis – Franz diffusion cell method: (1) Receiving solution: PBS buffer, 15 mL of receiving solution in the receiving chamber; (2) Diffusion cell: FITC solution, FITC-labeled collagen are tertiary embedded and physically mixed; 2 mL of each sample to be tested is added to the diffusion cell, and the diffusion cell is covered with a sealing film to prevent the solution from evaporating; (3) The stratum corneum of the pig skin faces upward, and the lower layer of skin is in just contact with the liquid in the receiving pool. The receiving pool is in contact with the skin to avoid air bubbles. (4) The water bath temperature of the diffuser is 37℃; the stirring speed is 350 rpm / min; (5) Collect 0.5 mL of sample from the receiving cell every 4 h and replenish the same volume of receiving solution at the same time, for a total of 24 h; (6) This experiment should be performed in the dark to prevent FITC fluorescence quenching.

[0048] Measurement of FITC fluorescence intensity: (1) Take 100 μL of receiving liquid from the receiving cells of each group above and put it into one well of a 96-well plate. Make three parallel wells for each group. (2) Protect from light and use a bioluminescence analyzer to detect the fluorescence intensity of the sample in the well.

[0049] Test results are as follows Figure 3 As shown, the composition of the present invention can promote transdermal absorption of collagen through three-stage encapsulation.

[0050] Performance Comparison of Example 6 and Alternative Materials

[0051] Experimental Materials and Methods: Three groups of samples were prepared: the present invention group (the rhodioloside / collagen / EGCG composition prepared in Example 1); the HP-β-cyclodextrin substitution group, in which β-cyclodextrin in Example 1 was replaced with HP-β-cyclodextrin, and the remaining preparation steps were the same as in Example 1; and the gelatin-chitosan substitution group, in which chitosan was replaced with gelatin, and the remaining preparation steps were the same as in Example 1. The following performance tests were performed on the three groups of samples: the encapsulation efficiency was determined by HPLC; the transdermal rate was determined by the Franz diffusion cell (ex vivo pig skin) experiment; the centrifugation stability was observed by centrifuging the samples at 4000 rpm for 15 min; and the Zeta potential at pH 6.5 was measured using a Zeta potential meter (Malvin Zetasizer Nano ZS, Malvin Panaco Ltd., UK). The results are shown in Table 5.

[0052] Table 5. Performance Comparison Results with Alternative Materials

[0053] Experimental results show that HP-β-cyclodextrin has an excessively large pore size (1.8 nm), which prevents it from tightly encapsulating EGCG, leading to easy leakage of EGCG in the system and thus reducing the encapsulation efficiency and transdermal penetration rate. Gelatin has an isoelectric point of pH 4.7-5.2, and at pH 6.5, it carries insufficient positive charge, which prevents it from effectively forming a stable positively charged shell, affecting the transdermal performance and stability of the carrier. The gelation phenomenon also indicates that gelatin cannot play an ideal protective and promoting role in this system.

[0054] Example 7: Efficacy Comparison with Commercially Available Competitors

[0055] Experimental Materials and Methods: Sample A was an essence matrix containing 5% (w / w) Bifida ferment lysate (the difference from Sample C is that it contains Bifida ferment lysate but not the composition of this invention). Sample B was an essence matrix containing 15% (w / w) Vitamin C and 1% (w / w) Vitamin E. Sample C was an anti-aging essence containing 5% (w / w) the composition of this invention. Sixty healthy women aged 35-50 years with similar skin conditions were selected as subjects through open recruitment and randomly divided into three groups of 20 each. Before the experiment, all subjects signed informed consent forms and discontinued using other anti-wrinkle, whitening, and moisturizing cosmetics for one week. Each group of subjects used the corresponding sample twice daily, morning and evening, with each application being 0.5g, for 28 days. Before the start of the experiment and after the 28-day experiment, parameters such as wrinkle depth, number and area of ​​UV spots, and transepidermal water loss were measured. One-way ANOVA was used for statistical analysis of the data. The results of the 28-day human test are shown in Table 6.

[0056] Table 6. Results of efficacy comparison with competing products on the market

[0057] Experimental results show that, compared with well-known competing products on the market, the rhodioloside / collagen / EGCG composition provided by this invention has more significant effects in reducing wrinkle depth, reducing UV spots, and reducing transdermal water loss, with statistically significant differences (P < 0.05 or P < 0.01).

[0058] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. A rhodioloside composition having a core-shell-shell structure, characterized in that: Its preparation method includes the following steps: 1) EGCG inclusion complex: β-cyclodextrin and EGCG were added to an appropriate amount of water at a mass ratio of (1.15-1.25):1 and ultrasonically treated to form a β-cyclodextrin-EGCG inclusion complex; 2) Liposome assembly: Weigh soybean lecithin and cholesterol, dissolve them in chloroform to form a homogeneous solution; weigh rhodioloside, dissolve it in the homogeneous solution, remove chloroform by rotary evaporation under vacuum and heating conditions, forming a soybean lecithin-cholesterol film on the container wall, then add the β-cyclodextrin-EGCG inclusion complex and PBS buffer to the container, hydrate, and form liposomes encapsulating rhodioloside and EGCG inclusion complex; 3) Shell loading: Weigh collagen and mix it evenly with chitosan solution to form a chitosan-collagen complex; add the complex dropwise to the liposomes, and through electrostatic adsorption, the chitosan-collagen complex coats the outer layer of the liposomes to form a core-shell-shell three-layer structure; centrifuge and purify to obtain a rhodioloside composition with a core-shell-shell structure.

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

1.

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

4. The rhodioloside composition having a core-shell-shell structure according to any one of claims 1 to 3, characterized in that: The mass ratio of the soybean phospholipids to the cholesterol is (65-75):(25-35).

5. The rhodioloside 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℃.

6. The rhodioloside 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℃.

7. The rhodioloside composition having a core-shell-shell structure according to any one of claims 1 to 3, characterized in that: The collagen has a molecular weight of 65-67 kDa, and the chitosan solution has a mass percentage concentration of 0.4%-0.6%; the ratio of collagen to chitosan solution is 45-55 mg: 8-12 mL.

8. The method for preparing the rhodioloside composition with a core-shell-shell structure according to claim 1, characterized in that: Includes the following steps: 1) EGCG inclusion complex: β-cyclodextrin and EGCG were added to an appropriate amount of water at a mass ratio of (1.15-1.25):1 and ultrasonically treated to form a β-cyclodextrin-EGCG inclusion complex; 2) Liposome assembly: Weigh soybean lecithin and cholesterol, dissolve them in chloroform to form a homogeneous solution; weigh rhodioloside, dissolve it in the homogeneous solution, remove chloroform by rotary evaporation under vacuum and heating conditions, forming a soybean lecithin-cholesterol film on the container wall, then add the β-cyclodextrin-EGCG inclusion complex and PBS buffer to the container, hydrate, and form liposomes encapsulating rhodioloside and EGCG inclusion complex; 3) Shell loading: Weigh collagen and mix it evenly with chitosan solution to form a chitosan-collagen complex; add the complex dropwise to the liposomes, and through electrostatic adsorption, the chitosan-collagen complex coats the outer layer of the liposomes to form a core-shell-shell three-layer structure; centrifuge and purify to obtain a rhodioloside composition with a core-shell-shell structure.

9. The use of the rhodioloside composition having a core-shell-shell structure according to claim 1 in the preparation of anti-aging / antioxidant cosmetics.

10. A cosmetic product, characterized in that: The rhodioloside composition having a core-shell-shell structure as described in any one of claims 1 to 7.

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