Preparation method and application of royal jelly acid double-wrapped body

Abstract

The application relates to a preparation method and application of royal jelly acid double-wrapped bodies, and belongs to the technical field of cosmetics. The royal jelly acid double-wrapped body prepared by the application is obtained by co-wrapping royal jelly acid by phospholipid lipids and hydroxypropyl-beta-cyclodextrin, and the double-wrapping system of the hydroxypropyl-beta-cyclodextrin and the liposome improves the loading capacity of the royal jelly acid, has good stability, can significantly improve the solubility of the royal jelly acid, and is convenient for application in cosmetics; compared with pure royal jelly acid, the royal jelly acid double-wrapped body prepared by the application has more excellent skin permeability and more excellent effect of improving the content of type IV collagen of skin, and can provide mechanical support for skin and maintain the young state of skin.

Description

TECHNICAL FIELD

[0001] The application relates to a preparation method and application of royal jelly acid double-wrapped bodies and belongs to the technical field of cosmetics. BACKGROUND

[0002] Royal jelly, also known as bee milk, is a yellowish-white, milky acid secretion secreted by the pharyngeal gland and the mandibular gland of a worker bee aged 5 to 15 days. Royal jelly acid, namely 10-hydroxy-2-decenoic acid (10-HDA), is an unsaturated fatty acid in royal jelly. Studies have shown that royal jelly acid has strong biological activity in terms of bacteriostasis, anti-inflammatory, antioxidant, whitening and the like, and has a wide application prospect. However, royal jelly acid has poor water solubility and oil solubility, and many application problems have been exposed in the daily experiment and product preparation process, such as small addition amount, difficulty in application in transparent water agent, poor stability and easy precipitation and the like.

[0003] In the prior art, patent application CN 117338613 A provides a composition for improving the solubility of royal jelly acid in water, which can effectively solve the application problem of royal jelly acid in transparent water agent and has excellent stability, but the composition contains a large amount of surfactant, which has the risk of strong irritation. At present, the cosmetic industry often solves the problems of poor solubility and stability of raw materials through liposome, microcapsule, cyclodextrin and the like wrapping technologies and the combination of various wrapping carriers, and can increase the transdermal absorption rate of active substances to a certain extent with the advantages of nano preparations. Patent application CN 117462450 A discloses a preparation method and application of royal jelly acid double-modified chitosan microspheres. The method can increase the solubility of royal jelly acid in water, but organic solvents are used in the preparation process, which increases the risk of environmental pollution and may cause certain organic solvent residues, which can easily cause skin irritation. In addition, the single delivery mode still has the problems of low drug loading, large addition amount in product application, increased application cost and instability. Patent application CN 115666602 A provides a colloidal system of royal jelly, which adopts double-wrapping technology, but the wrapped royal jelly is a water-soluble substance, and the content of royal jelly acid in the royal jelly is low, only 1.4-2.4%, which leads to low actual drug loading of royal jelly acid in the wrapping system, poor stability and compatibility, and is not conducive to subsequent use.

[0004] In the prior art, most of the double wrapping technology of liposome-cyclodextrin is to first use cyclodextrin to fully wrap the active substance, then use the cyclodextrin inclusion as a single substance, and then use the liposome to wrap, which is equivalent to liposome wrapping of water-soluble ingredients, and the wrapped ingredients only exist in the water phase. In addition, the active substance that is not wrapped in the first step of the cyclodextrin system is often in a precipitated state, and a solvent with high solubility for the ingredient needs to be used for subsequent liposome wrapping, which will extract the active ingredient from the cyclodextrin inclusion, resulting in an unstable system.

[0005] Therefore, the prior art still needs to be improved and developed. SUMMARY

[0006] In view of the shortcomings of the prior art, the application provides a preparation method and application of royal jelly acid double wrapping. The royal jelly acid double wrapping in the application is wrapped with phospholipid lipids and hydroxypropyl-beta-cyclodextrin, so that the wrapped royal jelly acid is not only distributed in the water phase (wrapped by hydroxypropyl-beta-cyclodextrin), but also distributed in the oil phase (the interlayer of the phospholipid bilayer), has a high drug loading capacity, and can effectively improve the solubility of royal jelly acid. The royal jelly acid double wrapping in the application can be flexibly applied to transparent water and cream products, has good stability, can promote the penetration of royal jelly acid, and improves the skin care effect.

[0007] The technical scheme of the application is as follows:

[0008] In a first aspect, the application provides a royal jelly acid double wrapping, which comprises raw materials: royal jelly acid, polyol phase, lipid phase, hydroxypropyl-beta-cyclodextrin, preservative component and ultrapure water.

[0009] According to a preferred embodiment of the application, the raw materials of the royal jelly acid double wrapping are as follows: royal jelly acid 0.1-5.0 parts, polyol phase 4.0-16.0 parts, lipid phase 1.0-5.0 parts, hydroxypropyl-beta-cyclodextrin 15.0-25.0 parts, preservative component 0.1-2.0 parts, and ultrapure water is added to 100 parts.

[0010] According to a preferred embodiment of the application, the polyol phase comprises one or more of 1,3-butanediol, 1,2-hexanediol, 1,3-propanediol and glycerol.

[0011] According to a preferred embodiment of the application, the lipid phase comprises one of hydrogenated lecithin or soybean phospholipid.

[0012] According to a preferred embodiment of the application, the preservative component comprises one or more of p-hydroxyacetophenone, phenoxyethanol, octanoyl hydroxamic acid, sodium benzoate, o-cymen-5-ol and chlorphenesin.

[0013] According to the application, preferably, the weight ratio of the lipid phase to the hydroxypropyl-beta-cyclodextrin is 1:5-1:15.

[0014] According to the application, preferably, the particle size of the royal jelly acid double-wrapped body is 50-200nm.

[0015] In the second aspect of the application, a preparation method of the royal jelly acid double-wrapped body is provided, comprising the following steps:

[0016] S1. Mixing the royal jelly acid, the ingredient with the antiseptic property and the polyhydric alcohol phase, and heating and stirring until the liquid is transparent to obtain phase A, and keeping warm;

[0017] S2. Continuously adding the lipid phase to the phase A, and stirring and dispersing uniformly to obtain phase B, and keeping warm;

[0018] S3. Dissolving the hydroxypropyl-beta-cyclodextrin in part of the ultrapure water to prepare a hydroxypropyl-beta-cyclodextrin aqueous solution, and heating to obtain phase C, and keeping warm;

[0019] S4. Under the shearing and homogenization condition, adding the phase C to the phase B, and continuously shearing and homogenizing;

[0020] S5. Stirring and cooling to 45-55℃, and adding the isothermal remaining ultrapure water under the stirring condition, and continuously stirring;

[0021] S6. Homogenizing the material obtained in step S5 for 2-3 times under high pressure until the material is clear, and the royal jelly acid double-wrapped body is obtained.

[0022] According to the application, preferably, in step S4, the time for continuously shearing and homogenizing is 3-7min.

[0023] According to the application, preferably, in step S6, the pressure for high-pressure homogenization is 750-900bar.

[0024] In the third aspect of the application, the above-mentioned royal jelly acid double-wrapped body is applied in cosmetics.

[0025] According to the application, preferably, the cosmetics include transparent water agent products or cream products.

[0026] Beneficial effects:

[0027] The royal jelly acid double-encapsulated complex prepared in this invention is obtained by co-encapsulating royal jelly acid with a phospholipid phase and hydroxypropyl-β-cyclodextrin. The double encapsulation system of hydroxypropyl-β-cyclodextrin and liposomes increases the loading capacity of royal jelly acid, exhibits good stability, and significantly improves the solubility of royal jelly acid, facilitating its application in cosmetics. Furthermore, the royal jelly acid double-encapsulated complex prepared in this invention has superior skin permeability and a superior effect on increasing the content of type IV collagen in the skin compared to pure royal jelly acid, providing mechanical support to the skin and maintaining a youthful appearance. Attached Figure Description

[0028] Figure 1 This is a particle size distribution diagram of the royal jelly acid double inclusions in Example 3.

[0029] Figure 2 This is a potential diagram of the royal jelly acid double inclusion complex in Example 3.

[0030] Figure 3 The images show the Raman spectra of the royal jelly acid double inclusions in Example 3. Figure a shows the distribution of royal jelly acid in the royal jelly acid double inclusions, indicated in red; Figure b shows the distribution of hydrogenated lecithin in the royal jelly acid double inclusions, indicated in green; Figure c shows the distribution of hydroxypropyl-β-cyclodextrin in the royal jelly acid double inclusions, indicated in orange; and Figure d shows a composite image of the distribution of royal jelly acid, hydrogenated lecithin, and hydroxypropyl-β-cyclodextrin in the royal jelly acid double inclusions.

[0031] Figure 4 The image shows an experiment on the irritation of the chicken embryo chorioallantoic membrane with royal jelly acid double-encapsulated material, as described in Example 3.

[0032] Figure 5 The figures show the Raman spectra of royal jelly acid in the gel samples of Example 4 and Comparative Example 9 permeating human skin. Figure a shows the Raman spectrum of royal jelly acid in the gel sample of Example 4 permeating human skin, and Figure b shows the Raman spectrum of royal jelly acid in the gel sample of Comparative Example 9 permeating human skin.

[0033] Figure 6 The bar chart shows the content of type IV collagen in isolated skin tissue under the action of pure royal jelly acid in Examples 3, Comparative Examples 6 and 8. Significance compared to the blank control group is indicated by #, with 0.01 < P < 0.05 indicated by # and P < 0.01 indicated by ##. Compared to the negative control group, significance is indicated by *, with 0.01 < P < 0.05 indicated by * and P < 0.01 indicated by **. Detailed Implementation

[0034] In order to better understand the technical solutions of the present application, the following further elaboration is made in combination with the examples, but the protection scope of the present application is not limited to the following examples, and the examples should not be regarded as limiting the protection scope of the present application.

[0035] The royal jelly acid used in the examples and comparative examples is derived from Shandong Furuida Biological Co., Ltd.; hydrogenated lecithin, soybean phospholipid and hydroxypropyl-β-cyclodextrin are all available from Shanghai Zhenya Biological Technology Co., Ltd. Other reagents and materials are all ordinary commercially available products unless otherwise specified. The raw material parts in the examples are all parts by weight.

[0036] Example 1

[0037] A royal jelly acid double-wrapped body, comprising raw materials: royal jelly acid 0.1 parts, phenoxyethanol 0.8 parts, 1,2-hexanediol 0.5 parts, glycerol 4.0 parts, hydrogenated lecithin 1.0 part, hydroxypropyl-β-cyclodextrin 15 parts, ultrapure water 78.6 parts, wherein the hydroxypropyl-β-cyclodextrin is dissolved in 7 parts of ultrapure water to prepare a hydroxypropyl-β-cyclodextrin aqueous solution.

[0038] The preparation method of the above-mentioned royal jelly acid double-wrapped body comprises the following steps:

[0039] S1. Mix royal jelly acid, phenoxyethanol, 1,2-hexanediol and glycerol, heat to 80°C and stir until the liquid is transparent to obtain phase A, and keep at 80°C;

[0040] S2. Continue to add hydrogenated lecithin to phase A, stir to disperse uniformly to obtain phase B, and keep at 80°C;

[0041] S3. Heat the hydroxypropyl-β-cyclodextrin aqueous solution to 80°C to obtain phase C, and keep at 80°C;

[0042] S4. Under the condition of shearing homogenization, add phase C to phase B, and continue shearing homogenization for 5 min;

[0043] S5. Stir to cool to 45-55°C, add isothermal remaining ultrapure water under stirring state, and continue stirring;

[0044] S6. Homogenize the material body obtained in step S5 under the condition of 800 bar for 2 times, until the material body is clear, to obtain the royal jelly acid double-wrapped body.

[0045] Example 2

[0046] A royal jelly acid double-encapsulated compound comprises the following raw materials: 2.5 parts royal jelly acid, 0.1 parts octanoyl hydroxamic acid, 0.5 parts 1,2-hexanediol, 15.0 parts 1,3-propanediol, 5.0 parts soybean lecithin, 25 parts hydroxypropyl-β-cyclodextrin, and 51.9 parts ultrapure water. The hydroxypropyl-β-cyclodextrin aqueous solution is prepared by dissolving the hydroxypropyl-β-cyclodextrin in 14 parts of ultrapure water beforehand.

[0047] The preparation method of the above-mentioned royal jelly acid double inclusions includes the following steps:

[0048] S1. Mix royal jelly acid, capryloyl hydroxamic acid, 1,2-hexanediol and 1,3-propanediol, heat to 60°C and stir until the liquid is transparent to obtain phase A, and keep warm at 60°C;

[0049] S2. Continue adding soybean lecithin to phase A, stir and disperse evenly to obtain phase B, and keep warm at 60℃;

[0050] S3. Heat the aqueous solution of hydroxypropyl-β-cyclodextrin to 60°C to obtain phase C, and keep it at 60°C.

[0051] S4. Under shear homogenization conditions, add phase C to phase B and continue shear homogenization for 3 min;

[0052] S5. Stir and cool to 45-55℃, add the remaining ultrapure water at the same temperature while stirring, and continue stirring;

[0053] S6. The material obtained in step S5 is homogenized twice under high pressure at 750 bar until the material is clear, thus obtaining the royal jelly acid double-encapsulated material.

[0054] Example 3:

[0055] A royal jelly acid double-encapsulated compound comprises the following raw materials: 5.0 parts royal jelly acid, 0.5 parts p-hydroxyacetophenone, 0.5 parts 1,2-hexanediol, 8.0 parts 1,3-butanediol, 3.6 parts hydrogenated lecithin, 20 parts hydroxypropyl-β-cyclodextrin, and 62.4 parts ultrapure water. The hydroxypropyl-β-cyclodextrin aqueous solution is prepared by dissolving the hydroxypropyl-β-cyclodextrin in 12 parts of ultrapure water beforehand.

[0056] The preparation method of the above-mentioned royal jelly acid double inclusions includes the following steps:

[0057] S1. Mix royal jelly acid, p-hydroxyacetophenone, 1,2-hexanediol and 1,3-butanediol, heat to 75°C and stir until the liquid is transparent to obtain phase A, and keep warm at 75°C.

[0058] S2. Continue adding hydrogenated lecithin to phase A, stir and disperse evenly to obtain phase B, and keep warm at 75℃;

[0059] S3. Heat the aqueous solution of hydroxypropyl-β-cyclodextrin to 75°C to obtain phase C, and keep it at 75°C.

[0060] S4. Under shear homogenization conditions, add phase C to phase B and continue shear homogenization for 7 min;

[0061] S5. Stir and cool to 45-55℃, add the remaining ultrapure water at the same temperature while stirring, and continue stirring;

[0062] S6. The material obtained in step S5 is homogenized twice under high pressure at 900 bar until the material is clear, thus obtaining the royal jelly acid double-encapsulated material.

[0063] Comparative Example 1:

[0064] A royal jelly acid double-encapsulated compound differs from the examples in that the weight ratio of the lipid phase to hydroxypropyl-β-cyclodextrin exceeds a specific range. The compound comprises the following raw materials: 5.0 parts royal jelly acid, 0.5 parts p-hydroxyacetophenone, 0.5 parts 1,2-hexanediol, 8.0 parts 1,3-butanediol, 6.0 parts hydrogenated lecithin, 26 parts hydroxypropyl-β-cyclodextrin, and 54 parts ultrapure water. The hydroxypropyl-β-cyclodextrin aqueous solution is prepared by dissolving the hydroxypropyl-β-cyclodextrin in 12 parts of ultrapure water beforehand.

[0065] The preparation method of the royal jelly acid double inclusion complex is the same as that in Example 3.

[0066] Comparative Example 2:

[0067] A royal jelly acid liposome, which differs from the examples in that it only encapsulates royal jelly acid with a phospholipid lipid phase to obtain a conventional liposome, includes the following raw materials: 5.0 parts royal jelly acid, 0.5 parts p-hydroxyacetophenone, 0.5 parts 1,2-hexanediol, 8.0 parts 1,3-butanediol, 8.0 parts soybean lecithin, and 78 parts ultrapure water.

[0068] The preparation method of the above-mentioned royal jelly acid liposomes includes the following steps:

[0069] Royal jelly acid, soybean lecithin, p-hydroxyacetophenone, 1,2-hexanediol, and 1,3-butanediol were mixed and heated to 75°C and stirred until the liquid was transparent. The mixture was then kept at 75°C. Under shear homogenization conditions, isothermal ultrapure water was added. The resulting mixture was homogenized twice under high pressure at 900 bar until the mixture became clear, thus obtaining royal jelly acid liposomes.

[0070] Comparative Example 3:

[0071] A royal jelly acid nanoemulsion differs from the examples in that it only encapsulates royal jelly acid with a phospholipid lipid phase to obtain a conventional nanoemulsion, comprising the following raw materials: 5.0 parts royal jelly acid, 0.5 parts p-hydroxyacetophenone, 0.5 parts 1,2-hexanediol, 8.0 parts 1,3-butanediol, 70 parts glycerol, 8.0 parts hydrogenated lecithin, and 8 parts ultrapure water.

[0072] The preparation method of the above-mentioned royal jelly acid nanoemulsion includes the following steps:

[0073] Royal jelly acid, p-hydroxyacetophenone, 1,2-hexanediol, 1,3-butanediol, and glycerol were mixed and heated to 75°C and stirred until the liquid became transparent to obtain phase A. The mixture was then kept at 75°C. Hydrogenated lecithin was added to phase A and stirred until it was dispersed evenly to obtain phase B. The mixture was then kept at 75°C. Under shear homogenization conditions, ultrapure water was added to phase B and shear homogenization was continued for 7 minutes. The resulting material was homogenized twice under high pressure at 900 bar until the material became clear, thus obtaining royal jelly acid nanoemulsion.

[0074] Comparative Example 4:

[0075] A royal jelly acid cyclodextrin inclusion complex differs from the examples in that it only encapsulates royal jelly acid with hydroxypropyl-β-cyclodextrin to obtain a conventional cyclodextrin inclusion complex. The complex comprises the following raw materials: 5.0 parts royal jelly acid, 0.5 parts p-hydroxyacetophenone, 0.5 parts 1,2-hexanediol, 40 parts 1,3-butanediol, 30 parts hydroxypropyl-β-cyclodextrin, and 24 parts ultrapure water. The hydroxypropyl-β-cyclodextrin aqueous solution is prepared by dissolving the hydroxypropyl-β-cyclodextrin in 24 parts of ultrapure water beforehand.

[0076] The preparation method of the above-mentioned royal jelly acid cyclodextrin inclusion complex includes the following steps:

[0077] Royal jelly acid, p-hydroxyacetophenone, 1,2-hexanediol, and 1,3-butanediol were mixed and heated to 75°C and stirred until the liquid became transparent to obtain phase A, which was then kept at 75°C. Hydroxypropyl-β-cyclodextrin aqueous solution was heated to 75°C to obtain phase B, which was then kept at 75°C. Under shear homogenization conditions, phase B was added to phase A, and shear homogenization was continued for 7 minutes. The resulting mixture was homogenized twice under high pressure at 900 bar until the mixture became clear, thus obtaining the royal jelly acid cyclodextrin inclusion complex.

[0078] Comparative Example 5:

[0079] A royal jelly acid double-encapsulated compound has the same raw material composition as Example 3, but the preparation method is different. It adopts a double-encapsulation process commonly found in literature and patents, including the following raw materials: 5.0 parts royal jelly acid, 0.5 parts p-hydroxyacetophenone, 0.5 parts 1,2-hexanediol, 8.0 parts 1,3-butanediol, 3.6 parts hydrogenated lecithin, 20 parts hydroxypropyl-β-cyclodextrin, and 62.4 parts ultrapure water. The hydroxypropyl-β-cyclodextrin aqueous solution is prepared by dissolving the hydroxypropyl-β-cyclodextrin in 62.4 parts of ultrapure water beforehand.

[0080] The preparation method of the above-mentioned royal jelly acid double inclusions includes the following steps:

[0081] Royal jelly acid, p-hydroxyacetophenone, 1,2-hexanediol, and 1,3-butanediol were mixed and heated to 75°C with stirring until the liquid became transparent to obtain phase A, which was then kept at 75°C. Hydroxypropyl-β-cyclodextrin aqueous solution was heated to 75°C to obtain phase B, which was then kept at 75°C. Under shear homogenization conditions, phase B was added to phase A, and shear homogenization was continued for 7 minutes. Hydrogenated lecithin was added and stirred until homogenized. The resulting material was homogenized twice under high pressure at 900 bar until the material became clear, thus obtaining the royal jelly acid double-encapsulated material.

[0082] Comparative Example 6:

[0083] A royal jelly colloidal system, as provided in patent application CN 115666602 A, is prepared using 5.0 parts of royal jelly raw material, wherein the royal jelly acid content is 0.08 parts, according to the preparation method of Example 2 described in patent application CN 115666602 A.

[0084] Comparative Example 7:

[0085] A royal jelly acid colloidal system, referring to the royal jelly colloidal system provided in patent application CN 115666602 A, replaces the raw material royal jelly with royal jelly acid, and uses 5.0 parts of royal jelly acid raw material, prepared according to the preparation method of Example 2 described in patent application CN 115666602 A.

[0086] Comparative Example 8:

[0087] A physical mixture of empty double-encapsulated material and pure royal jelly acid is provided, with a mixing mass ratio of empty double-encapsulated material to pure royal jelly acid of 19:1. The difference between the empty double-encapsulated material and the raw material in Example 3 is that it does not contain royal jelly acid, while the preparation process is the same as in Example 3.

[0088] Example 4 and Comparative Example 9:

[0089] Example 4 and Comparative Example 9 each provide a gel sample, wherein Example 4 contains the royal jelly acid double inclusion complex prepared in Example 3, and Comparative Example 9 contains pure royal jelly acid. The royal jelly acid content in Example 4 and Comparative Example 9 is the same, and their composition is shown in Table 1. They were prepared using conventional methods in the art. The above gel samples were used for in vivo human skin Raman permeation experiments.

[0090] Table 1. Raw material composition and ratio of gel samples from Example 4 and Comparative Example 9, unit: wt%.

[0091] Ingredients Example 4 Comparative Example 9 1,3-Butylene glycol 12 12 Royal jelly acid component 2 (double-wrapped body of royal jelly acid of Example 3) 0.1 (pure royal jelly acid) Carbomer 0.3 0.3 4-Hydroxyacetophenone 0.5 0.5 1,2-Hexanediol 0.5 0.5 Trometamol 0.24 0.24 Ultra-pure water 84.46 86.36

[0092] Experimental example:

[0093] I. Stability tests of samples from Examples 1-3 and Comparative Examples 1-5 and 7

[0094] 1. Experimental method: The samples prepared in Examples 1-3 and Comparative Examples 1-5 and 7 were placed under the following conditions for observation: 45℃, room temperature (RT), 4℃, -20℃, hot and cold cycling (-18℃, 4℃, 45℃, 24h at each temperature) and light (4500±500Lux). The observation period was 30 days.

[0095] 2. Experimental Results: The results are shown in Table 2. Samples from Examples 1-3 maintained good condition under room temperature, thermal cycling, 45℃, -20℃, light exposure, and 4℃ conditions, without precipitation or turbidity. They remained stable for 30 days under all six test conditions. Comparative Example 1 showed yellowing and a significant decrease in clarity under both 45℃ and thermal cycling conditions within the 30-day observation period, indicating that exceeding a certain range and ratio of hydrogenated lecithin and hydroxypropyl-β-cyclodextrin affects sample stability. Comparative Example 2 failed to emulsify uniformly, Comparative Example 3 showed significant precipitation at room temperature within 24 hours, and Comparative Example 4 showed precipitation under low-temperature (4℃, -20℃, thermal cycling) tests within 30 days. These results demonstrate that using single liposomes, nanoemulsions, or cyclodextrin inclusion complexes cannot stably encapsulate 5% royal jelly acid. Comparative Example 5 precipitated at room temperature within 24 hours, and the sample became turbid, indicating that the method of first encapsulating royal jelly acid with hydroxypropyl-β-cyclodextrin and then loading it as a water-soluble component into liposomes cannot support a large amount of pure royal jelly acid. Comparative Example 7 precipitated and separated after preparation, indicating that the colloidal system provided in patent application CN 115666602 A is not suitable for encapsulating pure royal jelly acid, let alone loading 5.0% royal jelly acid. The reason why the samples prepared in Examples 1-3 can remain stable for 30 days may be that the simultaneous encapsulation of royal jelly acid with phospholipid phase and hydroxypropyl-β-cyclodextrin in this invention ensures that the encapsulated royal jelly acid is not only encapsulated in hydroxypropyl-β-cyclodextrin but also widely distributed in the interlayer of the phospholipid bilayer, which is beneficial to the stability of the encapsulation system and can also overcome the limitations of using royal jelly acid.

[0096] Table 2. Stability test results of Examples 1-3 and Comparative Examples 1-5, 7

[0097]

[0098] Note: "√" indicates that the stability result is normal, and " / " indicates that observation will no longer continue after instability occurs.

[0099] II. Particle size and potential determination of sample in Example 3

[0100] 1. Experimental Methods: The particle size and distribution, polydispersity index (PDI), and zeta potential of the sample from Example 3 were measured using a BeNano 90Zeta nanoparticle size and zeta potential analyzer from Dandong Baite. The test angle was 90°, and the experimental temperature was 25°C. The sample was filtered through a 450nm filter membrane, placed in the sample cell, and equilibrated for 1 minute under the set instrument parameters. The measurement was repeated 3 times, and the data were recorded. Before measurement, the sample was diluted 10 times with ultrapure water to avoid interference from multiple scattering phenomena.

[0101] 2. Experimental results: such as Figure 1 andFigure 2 As shown, the particle size of the sample in Example 3 is in the range of 50-200 nm, with an average particle size of (66.0±2.0) nm, a Zeta potential of (-10.5±1.3) mV, and a polydispersity index of 0.258. The surface charge of the sample in Example 3 is a moderate negative ion, indicating that it has good stability, and the PDI<0.3 indicates that the system has reasonable size uniformity.

[0102] III. Raman spectral morphology of sample in Example 3

[0103] 1. Experimental Methods: Characteristic peaks of the two wall materials used in Example 3, hydrogenated lecithin and hydroxypropyl-β-cyclodextrin, were traced using Raman spectroscopy, and peaks showing inherent differences were selected for calibration. The degree of encapsulation of the active ingredients and the distribution of the encapsulating wall materials were analyzed using Raman imaging.

[0104] 2. Experimental Results: The results are as follows Figure 3 As shown, red, green, and orange represent the signals of royal jelly acid, hydrogenated lecithin, and hydroxypropyl-β-cyclodextrin, respectively. The results indicate that some royal jelly acid is encapsulated within the internal cavity of hydroxypropyl-β-cyclodextrin, while some royal jelly acid is dissolved in polyols and distributed within the palisade layer of the liposomes. Furthermore, some royal jelly acid-cyclodextrin inclusion complexes exist within the internal water cavities of the liposomes. The two systems are mutually integrated, maintaining stability.

[0105] IV. Safety Evaluation of Sample 3 in Example 3

[0106] 1. Experimental Methods: This study employed the reaction time method. Nine-day-old chicken embryos were candled, and the air cell location was marked on the eggshell surface. The marked portion of the eggshell was then peeled off using dental serrated forceps to expose the white egg membrane. A suitable amount of 0.9% NaCl solution was drawn using a pipette to moisten the egg membrane, and the solution was then poured out. The inner membrane was carefully removed with forceps, ensuring no damage to the vascular membrane. 0.3 mL of the test reagent (10% aqueous solution of the sample from Example 3) was added to the surface of the chorioallantoic membrane. The time to the onset of hemorrhage, vascular lysis, and coagulation within 5 minutes was recorded, and video and photographs were taken during this period.

[0107] 2. Experimental results: such as Figure 4 As shown, the test results of the 10% concentration of the royal jelly acid double-encapsulated aqueous solution in Example 3 showed that it was non-irritating.

[0108] V. Raman permeation experiment of royal jelly acid in samples of Example 4 and Comparative Example 9 on human skin

[0109] 1. Experimental Methods: Raman spectroscopy was used to trace the characteristic peaks of the samples from Example 4 and Comparative Example 9 applied to human skin, and characteristic peaks that differed from those on the skin itself were selected for calibration. Raman image analysis was used to determine the permeability of the active substance royal jelly acid in human skin after application of the samples from Example 4 and Comparative Example 9, thereby calculating the cumulative permeability of royal jelly acid in Example 4 and Comparative Example 9.

[0110] 2. Experimental results: as shown in Table 3 and Figure 5 As shown, the penetration effect of royal jelly acid in the sample of Example 4 is better than that in the sample of Comparative Example 9. When the royal jelly acid of Example 4 and Comparative Example 9 is applied to human skin for 10 hours, the cumulative penetration rate is 8.77% and 6.04%, respectively. The royal jelly acid in Example 4 continues to penetrate into each dermal layer within 1 hour, 2 hours, 4 hours, 6 hours, 8 hours and 10 hours, and the distribution is more uniform. This indicates that the royal jelly acid double encapsulation provided by the present invention has better penetration performance than pure royal jelly acid.

[0111] Table 3. Relative cumulative permeability of royal jelly acid in samples of Example 4 and Comparative Example 9

[0112]

[0113] VI. Effects of Samples from Example 3, Comparative Examples 6 and 8, and Pure Royal Jelly Acid on the Content of Type IV Collagen in Isolated Skin Tissue

[0114] 1. Experimental Methods: A skin photoaging model was established by irradiating isolated skin tissue with a combination of UVA and UVB rays. A blank control group and a negative control group (30 J / cm²) were included in the experiment. 2 UVA +50mJ / cm 2 UVB) and sample group (30J / cm) 2 UVA +50mJ / cm 2 UVB (Example 3 / Comparative Example 6 / Comparative Example 8 / Pure Royal Jelly Acid), 3 replicates per group. After culturing ex vivo skin tissue for 2 days, except for the blank control group, the other groups began irradiation and drug administration. The daily irradiation dose was UVA 30 J / cm². 2 and UVB 50mJ / cm 2During irradiation, the culture medium was discarded and replaced with PBS buffer. UVA irradiation was performed first, followed by UVB irradiation, for four consecutive days. After each irradiation, the culture medium was replaced with fresh medium, and surface drug administration was performed. Different groups of test solutions were added to the surface of the ex vivo skin tissue, with a volume of 2 μL. After four days of continuous irradiation, the ex vivo skin tissue was cultured for another three days, receiving only drug administration without irradiation. After culture, the surface of the ex vivo skin tissue was washed to remove any remaining test solution and residual liquid. The tissue was fixed with 4% paraformaldehyde, then embedded and sectioned. After dewaxing, COL-IV immunofluorescence staining was performed, and images were taken under a fluorescence microscope within 24 hours. Three images from different fields of view were acquired. The relative IOD of the target signal in the images was analyzed using Ipwin32 image analysis software, and the average value was calculated. Using the blank control group as a baseline, the relative IOD / area average was calculated to characterize the relative protein expression level. In this study, the test concentrations of Example 3, Comparative Example 6, Comparative Example 8, and pure royal jelly acid were 1.25% (royal jelly acid content was 0.0625%), stock solution (royal jelly acid content was approximately 0.08%), 1.25% (royal jelly acid content was 0.0625%), and 0.0625%, respectively. The diluent was EpiGrowth culture medium, and the test solutions for the blank control group and negative control group were EpiGrowth culture medium.

[0115] 2. Experimental Results: As a key component of the skin's basement membrane, type IV collagen acts as an "invisible scaffold" for the skin, maintaining its youthful appearance. (See Table 4 and...) Figure 6 As shown, after ultraviolet stimulation, the content of type IV collagen in the negative control group was significantly lower than that in the blank control group (P<0.01), proving the successful modeling of the skin photoaging model. After ultraviolet stimulation, compared with the negative control group, the content of type IV collagen was significantly increased after adding the samples of Example 3, Comparative Examples 6 and 8, and pure royal jelly acid to the photoaging model of isolated skin tissue (P<0.01). Among them, the increase rates of type IV collagen by the samples of Example 3, Comparative Examples 6 and 8, and pure royal jelly acid were 90.57%, 69.81%, 71.70%, and 67.92%, respectively. The above results show that the royal jelly acid double encapsulation in this invention can significantly increase the content of type IV collagen in the photoaging isolated skin model, provide mechanical support for the skin, maintain the youthful state of the skin, and the effect is better than the colloidal system of Comparative Example 6, the physical mixing system of Comparative Example 8, and pure royal jelly acid with the same encapsulation amount as the double encapsulation.

[0116] Table 4. Results of type IV collagen content in isolated skin tissue under different sample conditions

[0117] Group Relative IOD / area average value SD P-value Elevation rate Blank control 1.00 0.09 / / Negative control 0.53 0.07 0.002## / Example 3 1.01 0.05 0.001** 90.57％ Comparative Example 6 0.90 0.04 0.001** 69.81％ Comparative Example 8 0.91 0.03 0.001** 71.70％ Pure royal jelly acid 0.89 0.08 0.004** 67.92％

[0118] The royal jelly acid double-encapsulated compound prepared in this invention is a transparent liquid that can be flexibly applied to transparent aqueous and cream products. It has good stability and can promote the penetration of royal jelly acid, increase the content of type IV collagen, and enhance its skin care efficacy.

Claims

1. A royal jelly acid doublet, characterized by, The raw materials include royal jelly acid, polyol phase, lipid phase, hydroxypropyl-β-cyclodextrin, ingredients with preservative properties, and ultrapure water. The raw materials of the royal jelly acid double-encapsulated body are as follows by weight: royal jelly acid 5.0 parts, polyol phase 4.0-16.0 parts, lipid phase 1.0-5.0 parts, hydroxypropyl-β-cyclodextrin 15.0-25.0 parts, ingredients with preservative properties 0.1-2.0 parts, and ultrapure water to make up to 100.0 parts; wherein the weight ratio of the lipid phase to the hydroxypropyl-β-cyclodextrin is 1:5-1:15; the lipid phase includes one of hydrogenated lecithin or soybean phospholipid; the polyol phase includes one or more of 1,3-butanediol, 1,2-hexanediol, 1,3-propanediol, and glycerol; The royal jelly acid double-encapsulated body is prepared according to the following steps: S1. Mix the royal jelly acid, ingredients with preservative properties, and polyol phase, heat and stir until the liquid is transparent, obtain phase A, and keep warm, the temperature of the keeping warm is 60-80℃; S2. Continue to add the lipid phase to phase A, stir and disperse uniformly to obtain phase B, and keep warm, the temperature of the keeping warm is 60-80℃; S3. Dissolve the hydroxypropyl-β-cyclodextrin in part of the ultrapure water to prepare a hydroxypropyl-β-cyclodextrin aqueous solution, heat to obtain phase C, and keep warm, the temperature of the keeping warm is 60-80℃; S4. Under the condition of shear homogenization, add phase C to phase B, and continue shear homogenization, the time of the continued shear homogenization is 3-7 min; S5. Stir to cool to 45-55℃, add isothermal remaining ultrapure water under stirring, and continue stirring; S6. Homogenize the material obtained in step S5 at high pressure for 2-3 times, the pressure of the high-pressure homogenization is 750-900 bar, until the material is clear, and the royal jelly acid double-encapsulated body is obtained.

2. The royal jelly acid doublet of claim 1, wherein, The ingredients with preservative properties include one or more of p-hydroxyacetophenone, phenoxyethanol, octanoyl hydroxamic acid, sodium benzoate, o-cymen-5-ol, and chlorphenesin.

3. The royal jelly acid doublet of claim 1, wherein The particle size of the royal jelly acid double-encapsulated body is 50-200 nm.

4. The royal jelly acid double-encapsulated body of claim 1 is applied in cosmetics.

5. The use according to claim 4, wherein the compound is ###0002### The cosmetics include transparent lotion products or cream products. The cosmetics include transparent lotion products or cream products.

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