Preparation method and application of royal jelly acid double inclusion

Abstract

The invention relates to a preparation method and application of royal jelly acid double inclusion, and belongs to the technical field of cosmetics. The royal jelly acid double inclusion prepared by the invention is obtained by wrapping royal jelly acid together with a phospholipid lipid phase and hydroxypropyl-beta-cyclodextrin, and due to a double wrapping system of hydroxypropyl-beta-cyclodextrin and liposome, the loading capacity of the royal jelly acid is improved, the stability is good, the solubility of the royal jelly acid can also be remarkably improved, and the royal jelly acid double inclusion can be used for preparing the royal jelly acid. The application in cosmetics is facilitated; compared with a royal jelly acid pure product, the royal jelly acid double inclusion prepared by the invention has more excellent skin permeability and more excellent effect of increasing the content of IV-type collagen in the skin, provides mechanical support for the skin and maintains the young state of the skin.

Description

Technical Field

[0001] The invention relates to a preparation method and application of a royal jelly acid double inclusion body, belonging to the technical field of cosmetics. Background Art

[0002] Royal jelly, also known as royal jelly and bee milk, is a yellow-white, milky acidic secretion secreted by the hypopharyngeal gland and maxillary gland of worker bees from 5 to 15 days old. Royal jelly acid, also known as 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 antibacterial, anti-inflammatory, antioxidant, and whitening effects, and has broad application prospects. However, the water solubility and oil solubility of royal jelly acid are relatively poor, and many application difficulties are exposed in daily experiments and product preparation processes, such as small addition amounts, difficulty in application in transparent aqueous solutions, poor stability, and easy precipitation.

[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 aqueous solution and has excellent stability, but the composition contains a large amount of surfactant, which has the risk of high irritation. At present, the cosmetics industry often solves the problem of poor solubility and stability of raw materials through encapsulation technologies such as liposomes, microcapsules, cyclodextrins, and a combination of multiple encapsulation carriers, and can increase the transdermal absorption rate of active substances to a certain extent by taking advantage of the advantages of nano-preparations. Patent application CN 117462450A discloses a preparation method and application of chitosan microspheres double modified with royal jelly acid, which 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 there may be a certain amount of organic solvent residues, which can easily cause skin irritation. In addition, the single delivery mode still has the problems of low drug loading, large amount of addition during product application, increased application cost and instability. Patent application CN 115666602 A provides a colloidal system of royal jelly, which adopts double encapsulation technology. However, the encapsulated royal jelly is a water-soluble substance, and the royal jelly acid content in the royal jelly is low, only 1.4-2.4%, resulting in a low actual drug loading of royal jelly acid in the encapsulation system, and poor stability and compatibility, which is not conducive to subsequent use.

[0004] In the prior art, the double encapsulation technology of liposome-cyclodextrin mostly uses cyclodextrin to fully encapsulate the active substance first, and then uses liposome to encapsulate the cyclodextrin inclusion compound as a single substance. This process is equivalent to encapsulating the water-soluble components with liposomes, in which the encapsulated components only exist in the aqueous phase. It should also be considered that the active substances that are not encapsulated in the cyclodextrin system in the first step are often in a precipitated state. When encapsulating in liposomes later, a solvent with high solubility for the component needs to be used, and this practice will extract the active components in the cyclodextrin inclusion compound, resulting in system instability.

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

[0006] In view of the deficiencies in the prior art, the present invention provides a method for preparing and using a royal jelly acid double inclusion. The royal jelly acid double inclusion described in the present invention uses a phospholipid lipid phase and hydroxypropyl-β-cyclodextrin to simultaneously encapsulate royal jelly acid, so that the encapsulated royal jelly acid is not only distributed in the aqueous phase (encapsulated by hydroxypropyl-β-cyclodextrin), but also distributed in large quantities in the oil phase (the interlayer of the phospholipid bilayer), with a high drug loading, which can effectively improve the solubility of royal jelly acid. The royal jelly acid double inclusion described in the present invention can be flexibly applied to transparent aqueous solutions and cream products, has good stability, and can promote the penetration of royal jelly acid and enhance its skin care efficacy.

[0007] The technical solution of the present invention is as follows:

[0008] The first aspect of the present invention provides a royal jelly acid double inclusion body, comprising raw materials: royal jelly acid, a polyol phase, a lipid phase, hydroxypropyl-β-cyclodextrin, an ingredient with a preservative property and ultrapure water.

[0009] Preferably, according to the present invention, the raw materials of the royal jelly acid double inclusion body are as follows in parts by weight: 0.1-5.0 parts of royal jelly acid, 4.0-16.0 parts of polyol phase, 1.0-5.0 parts of lipid phase, 15.0-25.0 parts of hydroxypropyl-β-cyclodextrin, 0.1-2.0 parts of ingredients with preservative properties, and ultrapure water is supplemented to 100 parts.

[0010] Preferably according to the present invention, the polyol phase comprises one or more of 1,3-butanediol, 1,2-hexanediol, 1,3-propylene glycol and glycerol.

[0011] Preferably according to the present invention, the lipid phase comprises one of hydrogenated lecithin or soybean lecithin.

[0012] Preferably according to the present invention, the ingredients having antiseptic properties include one or more of p-hydroxyacetophenone, phenoxyethanol, caprylhydroxamic acid, sodium benzoate, o-cymene-5-ol, and chlorphenesin.

[0013] Preferably according to the present invention, the weight ratio of the lipid phase to hydroxypropyl-β-cyclodextrin is 1:5-1:15.

[0014] Preferably according to the present invention, the particle size of the royal jelly acid double inclusions is 50-200 nm.

[0015] The second aspect of the present invention provides a method for preparing royal jelly acid double inclusions, comprising the following steps:

[0016] S1. The royal jelly acid, the antiseptic component and the polyol are mixed, heated and stirred until the liquid is transparent to obtain phase A, and kept warm;

[0017] S2. Continue to add lipid phase to phase A, stir and disperse evenly to obtain phase B, and keep warm;

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

[0019] S4. Under shear homogenization conditions, add phase C to phase B and continue shearing and homogenizing;

[0020] S5. Cool down to 45-55°C with stirring, add the remaining ultrapure water at isothermal temperature while stirring, and continue stirring;

[0021] S6. The material obtained in step S5 is subjected to high pressure homogenization for 2-3 times until the material is clarified, thereby obtaining royal jelly acid double inclusions. Preferably, in steps S1, S2 and S3, the temperature of the insulation is 60-80°C.

[0022] Preferably according to the present invention, in step S4, the time for continuing shearing and homogenizing is 3-7 minutes.

[0023] Preferably according to the present invention, in step S6, the pressure of high pressure homogenization is 750-900 bar.

[0024] The third aspect of the present invention provides the use of the royal jelly acid double inclusions in cosmetics.

[0025] Preferably according to the present invention, the cosmetic comprises a transparent aqueous product or a cream product.

[0026] Beneficial effects:

[0027] The royal jelly acid double inclusions prepared by the present invention are obtained by co-encapsulating royal jelly acid with a phospholipid lipid phase and hydroxypropyl-β-cyclodextrin. The double encapsulation system of hydroxypropyl-β-cyclodextrin and liposomes increases the loading amount of royal jelly acid, has good stability, and can also significantly improve the solubility of royal jelly acid, making it easy to use in cosmetics; and the royal jelly acid double inclusions prepared by the present invention have better skin permeability than pure royal jelly acid, and have a better effect of increasing the content of type IV collagen in the skin, providing mechanical support for the skin and maintaining the youthfulness of the skin. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0029] Figure 2 This is the potential diagram of the royal jelly acid double inclusions of Example 3.

[0030] Figure 3 It is a Raman spectrum morphology diagram of the royal jelly acid double inclusions of Example 3; wherein, Figure a represents the distribution of royal jelly acid in the royal jelly acid double inclusions, represented by red; Figure b represents the distribution of hydrogenated lecithin in the royal jelly acid double inclusions, represented by green; Figure c represents the distribution of hydroxypropyl-β-cyclodextrin in the royal jelly acid double inclusions, represented by orange; Figure d represents a composite diagram of the distribution of royal jelly acid, hydrogenated lecithin and hydroxypropyl-β-cyclodextrin in the royal jelly acid double inclusions.

[0031] Figure 4 This is a picture of the chicken embryo chorioallantoic membrane irritation test of the royal jelly acid double inclusions of Example 3.

[0032] Figure 5 These are Raman spectra of the penetration of royal jelly acid in the gel samples of Example 4 and Comparative Example 9 on human skin; wherein, Figure a is a Raman spectra of the penetration of royal jelly acid in the gel sample of Example 4 on human skin, and Figure b is a Raman spectra of the penetration of royal jelly acid in the gel sample of Comparative Example 9 on human skin.

[0033] Figure 6 The bar graph is a graph of the type IV collagen content in the in vitro skin tissue under the action of the samples of Example 3 and Comparative Examples 6 and 8 and pure royal jelly acid; compared with the blank control group, the significance is represented by #, 0.01<P<0.05 is represented by #, and P<0.01 is represented by ##; compared with the negative control group, the significance is represented by *, 0.01<P<0.05 is represented by *, and P<0.01 is represented by **. DETAILED DESCRIPTION

[0034] In order to better understand the technical solution of the present invention, further elaboration is given below in conjunction with embodiments, but the protection scope of the present invention is not limited only to the following embodiments, and the embodiments should not be regarded as limiting the protection scope of the present invention.

[0035] The royal jelly acid used in the examples and comparative examples is from Shandong Freda Biological Co., Ltd.; hydrogenated lecithin, soybean lecithin and hydroxypropyl-β-cyclodextrin can all be purchased from Shanghai Zhina Biotechnology Co., Ltd. Unless otherwise specified, other reagents and materials are all common commercial products. The parts of raw materials in the examples are all parts by weight.

[0036] Embodiment 1:

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

[0038] The method for preparing the royal jelly acid double inclusions comprises the following steps:

[0039] S1. royal jelly acid, phenoxyethanol, 1,2-hexanediol and glycerin were mixed, heated to 80°C and stirred evenly until the liquid became transparent to obtain phase A, and kept at 80°C;

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

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

[0042] S4. Add phase C to phase B under shear homogenization conditions and continue shear homogenization for 5 minutes;

[0043] S5. Cool down to 45-55°C with stirring, add the remaining ultrapure water at isothermal temperature while stirring, and continue stirring;

[0044] S6. The material obtained in step S5 is homogenized twice under high pressure at 800 bar until the material is clarified, thereby obtaining a double inclusion of royal jelly acid.

[0045] Embodiment 2:

[0046] A royal jelly acid double inclusion body comprises raw materials: 2.5 parts of royal jelly acid, 0.1 parts of octanoylhydroxamic acid, 0.5 parts of 1,2-hexanediol, 15.0 parts of 1,3-propylene glycol, 5.0 parts of soybean lecithin, 25 parts of hydroxypropyl-β-cyclodextrin, and 51.9 parts of ultrapure water, wherein hydroxypropyl-β-cyclodextrin is dissolved in 14 parts of ultrapure water in advance to prepare a hydroxypropyl-β-cyclodextrin aqueous solution.

[0047] The method for preparing the royal jelly acid double inclusions comprises the following steps:

[0048] S1. royal jelly acid, octanoyl hydroxamic acid, 1,2-hexanediol and 1,3-propylene glycol are mixed, heated to 60°C and stirred evenly until the liquid becomes transparent to obtain phase A, and kept at 60°C;

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

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

[0051] S4. Add phase C to phase B under shear homogenization conditions and continue shear homogenization for 3 minutes;

[0052] S5. Cool down to 45-55°C with stirring, add the remaining ultrapure water at isothermal 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 clarified, thereby obtaining a double inclusion of royal jelly acid.

[0054] Embodiment 3:

[0055] A royal jelly acid double inclusion body comprises raw materials: 5.0 parts of royal jelly acid, 0.5 parts of p-hydroxyacetophenone, 0.5 parts of 1,2-hexanediol, 8.0 parts of 1,3-butanediol, 3.6 parts of hydrogenated lecithin, 20 parts of hydroxypropyl-β-cyclodextrin, and 62.4 parts of ultrapure water, wherein hydroxypropyl-β-cyclodextrin is dissolved in 12 parts of ultrapure water in advance to prepare a hydroxypropyl-β-cyclodextrin aqueous solution.

[0056] The method for preparing the royal jelly acid double inclusions comprises the following steps:

[0057] S1. royal jelly acid, p-hydroxyacetophenone, 1,2-hexanediol and 1,3-butanediol were mixed, heated to 75°C and stirred evenly until the liquid became transparent to obtain phase A, and kept at 75°C;

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

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

[0060] S4. Add phase C to phase B under shear homogenization conditions and continue shear homogenization for 7 minutes;

[0061] S5. Cool down to 45-55°C with stirring, add the remaining ultrapure water at isothermal 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 clarified, thereby obtaining a double inclusion of royal jelly acid.

[0063] Comparative Example 1:

[0064] A royal jelly acid double inclusion body, which is different from the embodiment in that the weight ratio of the lipid phase to hydroxypropyl-β-cyclodextrin exceeds a specific range, and includes the following raw materials: 5.0 parts of royal jelly acid, 0.5 parts of p-hydroxyacetophenone, 0.5 parts of 1,2-hexanediol, 8.0 parts of 1,3-butanediol, 6.0 parts of hydrogenated lecithin, 26 parts of hydroxypropyl-β-cyclodextrin, and 54 parts of ultrapure water, wherein hydroxypropyl-β-cyclodextrin is pre-dissolved in 12 parts of ultrapure water to prepare a hydroxypropyl-β-cyclodextrin aqueous solution.

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

[0066] Comparative Example 2:

[0067] A royal jelly acid liposome, which is different from the embodiment in that conventional liposomes are obtained by encapsulating royal jelly acid only with phospholipid lipid phase, and includes the following raw materials: 5.0 parts of royal jelly acid, 0.5 parts of p-hydroxyacetophenone, 0.5 parts of 1,2-hexanediol, 8.0 parts of 1,3-butanediol, 8.0 parts of soybean lecithin, and 78 parts of ultrapure water.

[0068] The preparation method of the royal jelly acid liposomes comprises the following steps:

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

[0070] Comparative Example 3:

[0071] A royal jelly acid nanoemulsion, which is different from the embodiment in that only phospholipid lipid phase is used to encapsulate royal jelly acid to obtain a conventional nanoemulsion, comprising the following raw materials: 5.0 parts of royal jelly acid, 0.5 parts of p-hydroxyacetophenone, 0.5 parts of 1,2-hexanediol, 8.0 parts of 1,3-butanediol, 70 parts of glycerol, 8.0 parts of hydrogenated lecithin, and 8 parts of ultrapure water.

[0072] The preparation method of the royal jelly acid nanoemulsion comprises the following steps:

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

[0074] Comparative Example 4:

[0075] A royal jelly acid-cyclodextrin inclusion compound, which differs from the embodiment in that a conventional cyclodextrin inclusion compound is obtained by encapsulating royal jelly acid only with hydroxypropyl-β-cyclodextrin, and comprises the following raw materials: 5.0 parts of royal jelly acid, 0.5 parts of p-hydroxyacetophenone, 0.5 parts of 1,2-hexanediol, 40 parts of 1,3-butanediol, 30 parts of hydroxypropyl-β-cyclodextrin, and 24 parts of ultrapure water, wherein hydroxypropyl-β-cyclodextrin is preliminarily dissolved in 24 parts of ultrapure water to prepare a hydroxypropyl-β-cyclodextrin aqueous solution.

[0076] The preparation method of the royal jelly acid cyclodextrin inclusion compound comprises the following steps:

[0077] Royal jelly acid, p-hydroxyacetophenone, 1,2-hexanediol and 1,3-butanediol were mixed, heated to 75°C and stirred evenly until the liquid was transparent to obtain phase A, and the mixture was kept at 75°C; the hydroxypropyl-β-cyclodextrin aqueous solution was heated to 75°C to obtain phase B, and the mixture was kept at 75°C; under shear homogenization conditions, phase B was added to phase A, and shear homogenization was continued for 7 minutes; the obtained material was homogenized under high pressure at 900 bar for 2 times until the material was clear, thereby obtaining the royal jelly acid cyclodextrin inclusion complex.

[0078] Comparative Example 5:

[0079] A royal jelly acid double inclusion body, whose raw material composition is the same as that of Example 3, but the preparation method is different. The double inclusion process commonly seen in literature and patents is adopted, including the raw materials: 5.0 parts of royal jelly acid, 0.5 parts of p-hydroxyacetophenone, 0.5 parts of 1,2-hexanediol, 8.0 parts of 1,3-butanediol, 3.6 parts of hydrogenated lecithin, 20 parts of hydroxypropyl-β-cyclodextrin, and 62.4 parts of ultrapure water, wherein hydroxypropyl-β-cyclodextrin is preliminarily dissolved in 62.4 parts of ultrapure water to prepare a hydroxypropyl-β-cyclodextrin aqueous solution.

[0080] The method for preparing the royal jelly acid double inclusions comprises the following steps:

[0081] Royal jelly acid, p-hydroxyacetophenone, 1,2-hexanediol and 1,3-butanediol were mixed, heated to 75°C and stirred evenly until the liquid was transparent to obtain phase A, and the mixture was kept at 75°C; the hydroxypropyl-β-cyclodextrin aqueous solution was heated to 75°C to obtain phase B, and the mixture was 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 evenly, and the obtained material was homogenized under high pressure at 900 bar for 2 times until the material was clear, thereby obtaining a double inclusion body of royal jelly acid.

[0082] Comparative Example 6:

[0083] A royal jelly colloid system is the royal jelly colloid system provided in patent application CN 115666602 A, which adopts 5.0 parts of royal jelly raw materials, wherein the royal jelly acid content is 0.08 parts, and is prepared according to the preparation method of Example 2 described in patent application CN 115666602 A.

[0084] Comparative Example 7:

[0085] A royal jelly acid colloid system is prepared by referring to the royal jelly colloid system provided in patent application CN 115666602 A, replacing the raw material royal jelly with royal jelly acid, using 5.0 parts of royal jelly acid raw material and following the preparation method of Example 2 described in patent application CN 115666602 A.

[0086] Comparative Example 8:

[0087] A physical mixture of an empty double inclusion and pure royal jelly acid, wherein the mixing mass ratio is empty double inclusion: pure royal jelly acid = 19:1, wherein the empty double inclusion differs from the raw material of Example 3 in that it does not contain royal jelly acid, and the preparation process is consistent with that of Example 3.

[0088] Example 4 and Comparative Example 9:

[0089] Example 4 and Comparative Example 9 respectively provide a gel sample, wherein Example 4 contains the royal jelly acid double inclusions 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 the composition ratio is shown in Table 1. The gel samples are prepared by conventional methods in the art. The above gel samples are used for the Raman penetration experiment of human skin in vivo.

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

[0091] Element Example 4 Comparative Example 9 1,3-Butanediol 12 12 Royal jelly acid ingredients 2 (Example 3 Royal Jelly Acid Double Inclusion) 0.1(Pure royal jelly acid) Carbomer 0.3 0.3 p-Hydroxyacetophenone 0.5 0.5 1,2-Hexanediol 0.5 0.5 Tromethamine 0.24 0.24 Ultrapure water 84.46 86.36

[0092] Experimental example:

[0093] 1. Stability Test of Examples 1 to 3 and Comparative Examples 1 to 5 and 7

[0094] 1. Experimental method: The samples prepared in Examples 1 to 3 and Comparative Examples 1 to 5 and 7 were placed at 45°C, room temperature (RT), 4°C, -20°C, hot and cold cycle (-18°C, 4°C, 45°C cycle, each temperature for 24h) and illumination (4500±500Lux) to observe the phenomena for a period of 30 days.

[0095] 2. Experimental results: The results are shown in Table 2. The samples of Examples 1 to 3 are in good condition at room temperature, hot and cold cycles, 45°C, -20°C, illumination and 4°C, without precipitation and turbidity, and can maintain a stable state within 30 days under 6 test conditions. In Comparative Example 1, the material turned yellow and the clarity decreased significantly under both 45°C and hot and cold cycle test conditions within the 30-day investigation period, indicating that hydrogenated lecithin and hydroxypropyl-β-cyclodextrin will affect the stability of the sample beyond a certain range and ratio. The material of Comparative Example 2 cannot be emulsified uniformly, Comparative Example 3 precipitates a large amount at room temperature within 24h, and Comparative Example 4 precipitates at low temperature (4°C, -20°C, hot and cold cycles) within 30 days. The above results show that it is impossible to stably encapsulate 5% royal jelly acid in the form of a single liposome, nanoemulsion or cyclodextrin inclusion compound. Comparative Example 5 was tested at room temperature within 24 hours and precipitated, and the material body became turbid, indicating that the royal jelly acid was first encapsulated with hydroxypropyl-β-cyclodextrin and then loaded into the liposome as a water-soluble component. The form of precipitating and stratifying after the preparation of Comparative Example 7 was completed, indicating that the colloid system provided in patent application CN 115666602 A is not suitable for encapsulating pure royal jelly acid, and it is even more impossible to load 5.0% of royal jelly acid. The reason why the samples prepared in Examples 1 to 3 can be stable within 30 days may be that the phospholipid lipid phase and hydroxypropyl-β-cyclodextrin are used in the present invention to simultaneously encapsulate royal jelly acid, so that the encapsulated royal jelly acid is not only encapsulated in hydroxypropyl-β-cyclodextrin, but also distributed in large quantities in the interlayer of the phospholipid bilayer, which is beneficial to the stability of the encapsulation system and can solve the use restrictions of royal jelly acid.

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

[0097]

[0098] Note: “√” means the stability result is normal, and “ / ” means no further observation will be made after the instability phenomenon occurs.

[0099] II. Particle size and potential measurement of samples in Example 3

[0100] 1. Experimental method: The particle size and distribution, polydispersity index (PDI), and Zeta potential of the sample in Example 3 were measured using Dandong Better's BeNano 90Zeta nanoparticle size and Zeta potential analyzer. The test angle was 90° and the experimental temperature was 25°C. The sample was filtered with a 450nm filter membrane, placed in a sample cell, balanced for 1 minute under the set instrument parameters, repeated 3 times, and the data was recorded. Ultrapure water was used to dilute 10 times before measurement to prevent the multiple scattering phenomenon of the sample from interfering with the measurement results.

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

[0102] 3. Raman spectrum morphology of the sample in Example 3

[0103] 1. Experimental method: Raman spectroscopy was used to track the characteristic peaks of the two wall materials, hydrogenated lecithin and hydroxypropyl-β-cyclodextrin, used in Example 3, and characteristic peaks with differences were selected for calibration. Raman images were used to analyze the degree of encapsulation of the active substance encapsulated sample and the distribution of the encapsulated wall material.

[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 show that part of the royal jelly acid is encapsulated in the internal cavity of hydroxypropyl-β-cyclodextrin, part of the royal jelly acid is dissolved in polyols and distributed in the palisade layer of the liposome, and part of the royal jelly acid-cyclodextrin inclusion complex exists in the inner water cavity of the liposome. The two systems fuse with each other to maintain stability.

[0105] IV. Safety evaluation of samples in Example 3

[0106] 1. Experimental method: This study adopts the reaction time method. The 9-day-old chicken embryos were examined by egg candles, and the position of the air chamber was marked on the eggshell surface; the marked eggshell part was peeled off with dental serrated curved forceps to expose the white egg membrane. After a proper amount of 0.9% NaCl solution was sucked with a pipette to moisten the egg membrane, the solution was poured out. Carefully remove the inner membrane with tweezers to ensure that the vascular membrane is not damaged. 0.3mL of the test agent (10% aqueous solution of the sample of Example 3) was drawn and dripped on the surface of the chorioallantoic membrane, and the time when bleeding, vascular melting, and coagulation began within 5 minutes was recorded, and video and photos were taken during the period.

[0107] 2. Experimental results: Figure 4 As shown, the test result of the royal jelly acid double inclusion aqueous solution of Example 3 with a concentration of 10% was non-irritating.

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

[0109] 1. Experimental method: The characteristic peaks of the samples of Example 4 and Comparative Example 9 applied to the human skin were tracked by Raman spectroscopy, and the characteristic peaks that were different from the skin itself were selected for calibration. The penetration ability of the active substance royal jelly acid in the samples of Example 4 and Comparative Example 9 was analyzed by Raman images, so as to calculate the cumulative penetration rate 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. The cumulative permeability of royal jelly acid in Example 4 and Comparative Example 9 when acting on human skin for 10 hours is 8.77% and 6.04%, respectively. The royal jelly acid in Example 4 continues to penetrate into each cortical layer within 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, and 10 hours, and is more evenly distributed, indicating that the royal jelly acid double inclusions provided by the present invention have more excellent permeability 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 the samples of Example 3 and Comparative Examples 6 and 8 and pure royal jelly acid on the content of type IV collagen in ex vivo skin tissue

[0114] 1. Experimental method: UVA+UVB was used to irradiate the ex vivo skin tissue to establish the skin photoaging model. The experiment set up a blank control group and a negative control group (30J / cm 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 the ex vivo skin tissue for 2 days, the remaining groups, except the blank control group, began to be irradiated and dosed. The daily irradiation dose was UVA 30 J / cm 2 and UVB 50mJ / cm 2, the culture medium was discarded during irradiation and replaced with PBS buffer, UVA irradiation was performed first, and then UVB irradiation was performed continuously for 4 days. Fresh culture medium was replaced after each irradiation, and surface drug administration was performed. The test liquids of different groups were added to the surface of the ex vivo skin tissue, and the added volume of the test liquid was 2 μL. After 4 days of continuous irradiation, the ex vivo skin tissue was cultured for another 3 days, and only drug administration was performed without irradiation. After the culture was completed, the test liquid remaining on the surface of the ex vivo skin tissue was cleaned and the residual liquid was removed. 4% paraformaldehyde was used for fixation, followed by tissue embedding and tissue sectioning. After the baked slices were dewaxed, COL-IV was subjected to conventional immunofluorescence staining, and photographed and observed under a fluorescence microscope within 24 hours. Three pictures of different fields of view were collected, and the target signals in the pictures were analyzed by relative IOD using Ipwin32 image analysis software, and the average value was calculated. The relative IOD / area average value was calculated based on the blank control group to characterize the relative expression of the protein. Among them, 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 about 0.08%), 1.25% (royal jelly acid content was 0.0625%) and 0.0625%, respectively. The diluent was EpiGrowth culture medium, and the test solution of the blank control group and the negative control group was EpiGrowth culture medium.

[0115] 2. Experimental results: As a key component of the skin basement membrane, type IV collagen is like the "invisible support" of the skin, maintaining the youthfulness of the skin. 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 that the skin photoaging model was successfully modeled; after ultraviolet stimulation, compared with the negative control group, after adding the samples of Example 3, Comparative Example 6, Comparative Example 8 and pure royal jelly acid to the in vitro skin tissue photoaging model, the content of type IV collagen increased significantly (P<0.01). Among them, the improvement rates of type IV collagen in Example 3, Comparative Example 6, Comparative Example 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 inclusions in the present invention can significantly increase the content of type IV collagen in the photoaging in vitro skin model, provide mechanical support for the skin, and 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 the pure royal jelly acid with the same amount of inclusions as the royal jelly acid double inclusions.

[0116] Table 4. Results of detection of type IV collagen content in ex vivo skin tissue under the action of different samples

[0117] Group Relative IOD / Area Average SD P-value Improvement 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％ Royal Jelly Acid Pure 0.89 0.08 0.004** 67.92％

[0118] The royal jelly acid double inclusions prepared in the present invention are transparent liquids, can be flexibly applied to transparent aqueous solutions and cream products, have 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 double inclusion, characterized in that: The invention comprises raw materials: royal jelly acid, a polyol phase, a lipid phase, hydroxypropyl-β-cyclodextrin, an ingredient with preservative properties and ultrapure water.

2. The royal jelly acid double inclusion according to claim 1, characterized in that: The raw materials of the royal jelly acid double inclusion body are as follows: 0.1-5.0 parts of royal jelly acid, 4.0-16.0 parts of polyol phase, 1.0-5.0 parts of lipid phase, 15.0-25.0 parts of hydroxypropyl-β-cyclodextrin, 0.1-2.0 parts of ingredients with antiseptic properties, and ultrapure water to 100.0 parts.

3. The royal jelly acid double inclusion according to claim 1, characterized in that: The polyol phase includes one or more of 1,3-butanediol, 1,2-hexanediol, 1,3-propylene glycol and glycerol.

4. The royal jelly acid double inclusion according to claim 1, characterized in that: The lipid phase includes one of hydrogenated lecithin or soybean lecithin.

5. The royal jelly acid double inclusion according to claim 1, characterized in that: The ingredients with antiseptic properties include one or more of p-hydroxyacetophenone, phenoxyethanol, caprylhydroxamic acid, sodium benzoate, o-cymene-5-ol, and chlorphenesin.

6. The royal jelly acid double inclusion according to claim 1, characterized in that: The weight ratio of the lipid phase to hydroxypropyl-β-cyclodextrin is 1:5-1:

15.

7. The royal jelly acid double inclusion according to claim 1, characterized in that: The particle size of the royal jelly acid double inclusions is 50-200 nm.

8. A method for preparing the royal jelly acid double inclusions according to claim 1, characterized in that: The steps include: S1. The royal jelly acid, the antiseptic component and the polyol are mixed, heated and stirred until the liquid is transparent to obtain phase A, and kept warm; S2. Continue to add lipid phase to phase A, stir and disperse evenly to obtain phase B, and keep warm; S3. dissolving hydroxypropyl-β-cyclodextrin in part of ultrapure water to prepare a hydroxypropyl-β-cyclodextrin aqueous solution, heating to obtain phase C, and keeping warm; S4. Under shear homogenization conditions, add phase C to phase B and continue shearing and homogenizing; S5. Cool down to 45-55°C with stirring, add the remaining ultrapure water at isothermal temperature while stirring, and continue stirring; S6. The material obtained in step S5 is subjected to high pressure homogenization for 2-3 times until the material is clarified, thereby obtaining a double inclusion body of royal jelly acid.

9. The preparation method according to claim 8, characterized in that: Meet one or more of the following conditions: i. In steps S1, S2 and S3, the insulation temperature is 60-80°C; ii. In step S4, the shearing and homogenization are continued for 3-7 minutes; iii. In step S6, the pressure of high-pressure homogenization is 750-900 bar.

10. Use of the royal jelly acid double inclusions according to claim 1 in cosmetics; Preferably, the cosmetic comprises a transparent aqueous product or a cream product.

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