Royal jelly acid nano-liposome with antioxidant and anti-inflammatory effects as well as preparation method and application of royal jelly acid nano-liposome

Abstract

The invention discloses royal jelly acid nano-liposome with antioxidant and anti-inflammatory effects as well as a preparation method and application of the royal jelly acid nano-liposome, and belongs to the technical field of liposome preparation. The royal jelly acid nano-liposome with the antioxidant and anti-inflammatory effects, provided by the invention, comprises the following raw materials in parts by weight: 5-10 parts of lipid, 2-16 parts of an emulsifier, 1-10 parts of a co-emulsifier, 1-5 parts of a stabilizer, 5-10 parts of royal jelly acid, 0.5-1 part of epigallocatechin gallate and 2-10 parts of water. Through the synergistic effect of a plurality of molecular pathways between royal jelly acid and epigallocatechin gallate, expression of pro-inflammatory cytokines and generation of inflammatory mediators can be reduced, meanwhile, active oxygen is removed, inflammatory response caused by oxidative stress is relieved, cytotoxicity is reduced, and the anti-inflammatory effect is achieved. Therefore, the royal jelly acid nano-liposome is endowed with excellent product stability, oxidation resistance and inflammation resistance, and has wide application in preparation of skin care products.

Description

Technical Field

[0001] The present application belongs to the technical field of liposome preparation, and in particular, relates to a royal jelly acid nanoliposome having both anti-oxidation and anti-inflammatory effects, and a preparation method and application thereof. Background Art

[0002] Royal jelly acid (10-HDA) is an unsaturated fatty acid mainly found in royal jelly. It has multiple biological activities and shows great application potential in the fields of medicine, health care and cosmetics. However, a large amount of single royal jelly acid is required to achieve significant antioxidant and anti-inflammatory effects, which will pose a potential risk to human health and increase production costs. Epigallocatechin Gallate (EGCG) is one of the main catechins in green tea and is widely used in cosmetics due to its powerful antioxidant and anti-inflammatory properties. However, the single use of epigallocatechin gallate has certain cytotoxicity, which affects its wide application. Summary of the invention

[0003] The present application discloses a royal jelly acid nanoliposome having both anti-oxidation and anti-inflammatory effects, and a preparation method and application thereof, aiming to solve the technical problems of the existing application of royal jelly acid liposomes in the field of cosmetics.

[0004] In order to achieve the above purpose, the technical solution of this application is:

[0005] The first aspect of the present application provides a royal jelly acid nanoliposome with both anti-oxidation and anti-inflammatory effects, and its components, calculated by weight of raw materials, include:

[0006] 5-10 parts of lipids,

[0007] Emulsifier 2-16 parts,

[0008] 1-10 parts of auxiliary emulsifier,

[0009] Stabilizer 1-5 parts,

[0010] 5-10 parts of royal jelly acid,

[0011] Epigallocatechin gallate 0.5-1 part,

[0012] 2-10 parts water.

[0013] In combination with the first aspect, preferably, the components thereof include, by weight of the raw materials:

[0014] 5-8 parts of lipids,

[0015] Emulsifier 2-10 parts,

[0016] 1-8 parts of auxiliary emulsifier,

[0017] 1-3 parts of stabilizer,

[0018] 5-8 parts of royal jelly acid,

[0019] Epigallocatechin gallate 0.5-0.8 parts,

[0020] 3-8 parts water.

[0021] In combination with the first aspect, preferably, the lipid is one or more of lecithin, caprylic / capric triglyceride, monostearate glyceryl, butyloctanol salicylate, and acetylated monoglyceride;

[0022] And / or, the emulsifier is one or more of polyglyceryl 10 stearate, PEG-8 caprylic / capric glyceride, sucrose stearate, cetearyl olivate, sorbitan olivate, laureth-23, PEG-4 olivate, steareth-21, palmityl alcohol, PEG-75 stearate, glyceryl stearate, steareth-20, cetyl alcohol, cetyl alcohol ether-20, and steareth-20.

[0023] In combination with the first aspect, preferably, the co-emulsifier is one or more of glycerol, 1,3-propylene glycol, 1,2-pentanediol ethoxydiglycol, olive oil PEG-7 esters, polyglyceryl-3 diisostearate, polyglyceryl-6 isostearate, polyethylene glycol-8, polyethylene glycol-12, polyethylene glycol-40, and polyethylene glycol-400.

[0024] In combination with the first aspect, preferably, the stabilizer is one or more of 1,3-butanediol, hydrogenated olive oil, olive oil, unsaponifiable matter of olive oil, cetyl palmitate, sorbitan palmitate, and sorbitan olivate.

[0025] In combination with the first aspect, preferably, the average particle size distribution of the royal jelly acid nanoliposomes having both antioxidant and anti-inflammatory effects is 100-200 nm.

[0026] The second aspect of the present application provides a method for preparing the royal jelly acid nanoliposomes having both anti-oxidation and anti-inflammatory effects as described in the first aspect, the method comprising:

[0027] The lipid, royal jelly acid and epigallocatechin gallate are dissolved in a solvent and evaporated under reduced pressure to obtain phase A;

[0028] The emulsifier, co-emulsifier and stabilizer are mixed and added into water to obtain phase B;

[0029] Phase A and phase B are mixed, stirred evenly, subjected to high-pressure homogenization treatment, and the pH value is adjusted to obtain the royal jelly acid nanoliposomes having both antioxidant and anti-inflammatory effects.

[0030] In combination with the second aspect, preferably, the conditions of the high-pressure homogenization treatment are: pressure of 20-100 MPa, temperature of 40-80° C., and number of cycles of 1-5 times.

[0031] In combination with the second aspect, preferably, the adjusted pH value is 6.5-7.0.

[0032] The third aspect of the present application provides the use of royal jelly acid nanoliposomes with antioxidant and anti-inflammatory effects prepared by the preparation method of the second aspect in the preparation of skin care products.

[0033] Compared with the prior art, the advantages or beneficial effects of the embodiments of the present application include at least:

[0034] The royal jelly acid nanoliposomes with both antioxidant and anti-inflammatory effects provided by the present application are prepared according to raw materials including lipids, emulsifiers, co-emulsifiers, stabilizers, water, royal jelly acid and epigallocatechin gallate. On the one hand, it can give nanoliposomes excellent product stability, reduce the average particle size, enhance contact with cells, and ensure the concentration of active substances within a certain time range; on the other hand, through the synergistic effect of multiple molecular pathways between royal jelly acid and epigallocatechin gallate, it can not only inhibit the activation of NF-κB pathway, reduce the expression of pro-inflammatory cytokines, but also jointly interfere with MAPK signaling pathways, reduce the generation of inflammatory mediators, and at the same time, remove reactive oxygen, reduce the inflammatory response caused by oxidative stress, and thus greatly improve the anti-inflammatory effect of liposomes; thirdly, through the compound use of royal jelly acid and epigallocatechin gallate, it can not only greatly reduce the dosage of a single raw material, but also reduce cytotoxicity, and at the same time greatly improve the antioxidant, anti-inflammatory and whitening effects, and has a wide range of market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments recorded in the present application, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0036] Figure 1 IC of positive control VC in the ABTS free radical scavenging experiment of royal jelly acid nanoliposomes with both antioxidant and anti-inflammatory effects provided in the examples of the present application 50 picture;

[0037] Figure 2 A statistical graph showing the results of ABTS free radical scavenging rate of royal jelly acid nanoliposomes with both anti-oxidation and anti-inflammatory effects provided in the examples of the present application;

[0038] Figure 3A statistical chart of the results of a cell anti-inflammatory experiment of royal jelly acid nanoliposomes with both anti-oxidation and anti-inflammatory effects provided in the examples of the present application;

[0039] Among them, compared with the LPS group, *P<0.05, **P<0.01, ***P<0.005, ****P<0.0001;

[0040] Figure 4 A schematic diagram of the in vivo cytotoxicity results of royal jelly acid nanoliposomes with both anti-oxidation and anti-inflammatory effects provided in the examples of the present application;

[0041] Among them, compared with the blank group, *P<0.05, **P<0.01, ***P<0.005, ****P<0.0001;

[0042] Figure 5 A schematic diagram of the results of a melanoma cell inhibition experiment of royal jelly acid nanoliposomes with both anti-oxidation and anti-inflammatory effects provided in an example of the present application;

[0043] Figure 6 This is a statistical graph showing the results of the total hemoglobin area reduction rate of the royal jelly acid nanoliposomes with antioxidant and anti-inflammatory effects provided in the examples of the present application. DETAILED DESCRIPTION

[0044] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0045] In the following description of this embodiment, the term "and / or" is used to describe the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, B exists alone, and A and B exist at the same time. A and B can be singular or plural. The character " / " generally indicates that the associated objects before and after are in an "or" relationship.

[0046] In the following description of this embodiment, the term "at least one" refers to one or more, and "plurality" refers to two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b or c", or "at least one of a, b and c", can all represent: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

[0047] Those skilled in the art should understand that in the following description of the embodiments of the present application, the order of serial numbers does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0048] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "an" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0049] Unless otherwise specified, all raw materials and reagents used in the examples of the present application are purchased on the market or prepared according to conventional methods well known to those skilled in the art.

[0050] In the first aspect, the present invention provides a royal jelly acid nanoliposome with both anti-oxidation and anti-inflammatory effects, wherein the components thereof include, by weight of raw materials: 5-10 parts of lipids,

[0051] Emulsifier 2-16 parts,

[0052] 1-10 parts of auxiliary emulsifier,

[0053] Stabilizer 1-5 parts,

[0054] 5-10 parts of royal jelly acid,

[0055] Epigallocatechin gallate 0.5-1 part,

[0056] 2-10 parts water.

[0057] Among them, on the one hand, it can give nanoliposomes excellent product stability, reduce the average particle size, enhance contact with cells, and ensure the concentration of active substances within a certain time range; on the other hand, through the synergistic effect of multiple molecular pathways between royal jelly acid and epigallocatechin gallate, it can not only inhibit the activation of the NF-κB pathway and reduce the expression of proinflammatory cytokines, but also jointly interfere with the MAPK signaling pathway, reduce the production of inflammatory mediators, and at the same time, remove reactive oxygen and reduce the inflammatory response caused by oxidative stress, thereby greatly enhancing the anti-inflammatory effect of liposomes; thirdly, through the combined use of royal jelly acid and epigallocatechin gallate, not only can the dosage of a single raw material be greatly reduced, but also the cytotoxicity can be reduced, and at the same time, the antioxidant, anti-inflammatory and whitening effects can be greatly improved, which has broad market application prospects.

[0058] It should be noted that royal jelly acid inhibits tyrosinase activity and reduces the production of melanin, thereby achieving a whitening effect. EGCG neutralizes free radicals through antioxidant effects, preventing skin oxidative damage and pigmentation caused by ultraviolet rays. In addition, EGCG can inhibit the activity of tyrosinase and further reduce the formation of melanin. When the two are used in combination, royal jelly acid and EGCG jointly inhibit melanin production through dual pathways, and the antioxidant effect of EGCG enhances the skin's defense against the external environment, thereby achieving a more significant whitening effect.

[0059] It should be noted that the average particle size distribution of the royal jelly acid nanoliposomes is 100-200nm. First, royal jelly acid and epigallocatechin gallate (EGCG) can inhibit the activation of the NF-κB pathway, reduce the expression of proinflammatory cytokines such as TNF-α, IL-6 and IL-1β, and synergistically inhibit the inflammatory response. Secondly, they can jointly interfere with the MAPK signaling pathway, inhibit the phosphorylation of ERK, JNK and p38 MAPK, thereby reducing the generation of inflammatory mediators. In addition, both have antioxidant properties, can scavenge reactive oxygen species (ROS), and reduce the inflammatory response caused by oxidative stress. It can also inhibit the activity of cyclooxygenase (COX-2) and lipoxygenase (LOX), and reduce the generation of inflammatory mediators such as prostaglandins and leukotrienes. Finally, EGCG enhances the anti-inflammatory ability of cells by regulating the AMPK signaling pathway, and royal jelly acid further relieves inflammation by promoting the apoptosis of inflammatory cells. In addition, both royal jelly acid and epigallocatechin gallate can inhibit tyrosinase activity, while epigallocatechin gallate can also scavenge free radicals and reduce UV-induced skin oxidative damage and pigmentation. Therefore, royal jelly acid and EGCG work synergistically through multiple pathways to significantly improve anti-inflammatory and whitening effects.

[0060] It should be noted that the present application does not impose any particular limitation on the selected solvent, as long as the lipids, royal jelly acid, and epigallocatechin gallate can be completely dissolved in the solvent to form a uniform and stable oil phase solution, and ether is more preferred.

[0061] In the embodiment of the present application, the emulsifier is preferably one or more of polyglycerol 10 stearate, PEG-8 caprylic / capric glyceride, sucrose stearate, cetearyl olive oil ester, sorbitan olive oil ester, laureth-23, PEG-4 olive oil ester, steareth-21, palmityl alcohol, PEG-75 stearate, glyceryl stearate, steareth-20, hexyl alcohol, hexyl alcohol ether-20, and steareth-20. Among them, through the selection of these emulsifiers, the oil phase and the water phase can be effectively mixed together, so that each component forms a stable and uniform emulsion, maintaining the stability of the product, and at the same time, these emulsifiers are mild and non-irritating, environmentally friendly, and suitable for large-scale application.

[0062] In the embodiment of the present application, the co-emulsifier is preferably one or more of glycerol, 1,3-propylene glycol, 1,2-pentanediol ethoxydiglycol, olive oil PEG-7 esters, polyglyceryl-3 diisostearate, polyglyceryl-6 isostearate, polyethylene glycol-8 (PEG-8), polyethylene glycol-12 (PEG-12), polyethylene glycol-40 (PEG-40), and polyethylene glycol-400 (PEG-400). Among them, through the selection of the co-emulsifier, an interface barrier can be formed on the surface of the droplet, delaying the migration of the monomer from the small droplet to the large droplet, so that it forms a smaller droplet, thereby improving the stability of the emulsion system.

[0063] In the embodiment of the present application, the stabilizer is preferably one or more of 1,3-butylene glycol, hydrogenated olive oil, olive oil, unsaponifiable olive oil, cetyl palmitate, sorbitan palmitate, and sorbitan olivate. Among them, through the selection of these stabilizers, in practical applications, the emulsification stabilization effect is good, the stabilization system effect is significant, the source is natural, and it has a certain moisturizing and absorption promoting effect.

[0064] The second aspect of the present application provides a method for preparing the royal jelly acid nanoliposomes having both anti-oxidation and anti-inflammatory effects as described in the first aspect, the method comprising:

[0065] The lipid, royal jelly acid and epigallocatechin gallate are dissolved in a solvent and evaporated under reduced pressure to obtain phase A;

[0066] The emulsifier, co-emulsifier and stabilizer are mixed and added into water to obtain phase B;

[0067] Phase A is added to phase B and mixed, stirred evenly, subjected to high-pressure homogenization, and the pH value is adjusted to obtain the royal jelly acid nanoliposomes with both antioxidant and anti-inflammatory effects.

[0068] It should be noted that the present application adopts an oil phase formed by lipids, royal jelly acid and epigallocatechin gallate and an aqueous phase formed by an emulsifier, an emulsifier and a stabilizer, and the process of mixing the oil phase and the aqueous phase for pre-emulsification can increase the stability of the effective ingredients of the liposome and the soothing effect. After high-pressure homogenization treatment, the oil phase and the aqueous phase are mixed more fully through high-speed impact, shearing and cavitation, which can effectively encapsulate the active substance, so that the active substance exists stably in the lipid system, giving the liposome excellent comprehensive performance. At the same time, the particle size distribution of the prepared nanoliposome can be made more uniform.

[0069] In the embodiment of the present application, the conditions of the high-pressure homogenization treatment are preferably: the pressure is preferably 20-100MPa, for example, 20MPa, 50MPa, 80MPa, 90MPa, 100MPa or any pressure within the range; the temperature is preferably 40-80°C, for example, 40°C, 50°C, 60°C, 70°C, 80°C or any temperature within the range; the number of cycles is preferably 1-5 times, for example, 1 time, 3 times, 5 times or any number within the range. Among them, by controlling the pressure, temperature and time of the high-pressure homogenization treatment, the raw materials can be sheared, impacted and homogenized under high pressure, and the liposome components can be evenly dispersed. At the same time, the particle size distribution of the liposomes can be adjusted, thereby improving the stability of the liposomes and product performance.

[0070] The third aspect of the present application provides the use of the royal jelly acid nanoliposomes with both antioxidant and anti-inflammatory effects prepared by the preparation method described in the second aspect in the preparation of skin care products. Among them, based on the above royal jelly acid nanoliposomes having excellent product stability, antioxidant, anti-inflammatory, whitening effects and no cytotoxicity, it improves the pathological microenvironment of the skin and promotes the healing of wounds, thereby giving excellent product competitiveness.

[0071] The technical solution of the present application will be further described below in conjunction with specific embodiments.

[0072] Example 1

[0073] This embodiment provides a method for preparing A1-royal jelly acid nanoliposomes having both anti-oxidation and anti-inflammatory effects, which specifically comprises:

[0074] S101: Dissolve 5 parts of lecithin in 200 parts of ether, stir thoroughly to dissolve, add 5 parts of royal jelly acid and 0.5 parts of EGCG, and perform ultrasound at 200 W power for 5 min to obtain an emulsion. Heat the emulsion to 45° C. in a water bath, and remove the ether by evaporation under reduced pressure to obtain phase A1;

[0075] S102: Heat 5 parts of polyglycerol 10 stearate, 1 part of glycerol, and 1 part of hydrogenated olive oil to 80° C., add them into 100 parts of deionized water, stir and mix them evenly, and obtain phase B1;

[0076] S103: Add phase A1 to phase B1, mix, homogenize and shear, with a shear speed of 10,000 rpm and a shear time of 10 min. High-pressure homogenization is performed at a pressure of 100 MPa and a temperature of 80° C. for 3 cycles. A pH regulator is added to adjust the pH value to 6.5-7, and the mixture is cooled to room temperature to obtain A1-royal jelly acid nanoliposomes with antioxidant and anti-inflammatory effects.

[0077] Example 2

[0078] This embodiment provides a method for preparing A2-royal jelly acid nanoliposomes having both anti-oxidation and anti-inflammatory effects, which specifically comprises:

[0079] S201: 8 parts of glyceryl monostearate were dissolved in 200 parts of ether, and the mixture was fully stirred and dissolved. 8 parts of royal jelly acid and 0.8 parts of EGCG were added, and ultrasound was performed at 200 W power for 5 min to obtain an emulsion. The emulsion was heated to 45° C. in a water bath, and the ether was removed by evaporation under reduced pressure to obtain phase A2;

[0080] S202: 10 parts of sucrose stearate, 1 part of 1,2-pentanediol ethoxydiglycol, and 5 parts of olive oil are heated to 80° C., added into 100 parts of deionized water, stirred and mixed evenly, to obtain phase B2;

[0081] S203: Add phase A2 to phase B2, mix, homogenize and shear, with a shear speed of 10,000 rpm and a shear time of 10 min. High-pressure homogenization is performed at a pressure of 100 MPa and a temperature of 80°C for 3 cycles. A pH adjuster is added to adjust the pH value to 6.5-7, and the mixture is cooled to room temperature to obtain A2-royal jelly acid nanoliposomes with antioxidant and anti-inflammatory effects.

[0082] Example 3

[0083] This embodiment provides a method for preparing A3-royal jelly acid nanoliposomes having both anti-oxidation and anti-inflammatory effects, which specifically comprises:

[0084] S301: Dissolve 10 parts of lecithin in 200 parts of ether, stir thoroughly to dissolve, add 10 parts of royal jelly acid and 1 part of EGCG, and perform ultrasound at 200W power for 5 minutes to obtain an emulsion, heat the emulsion in a water bath to 45°C, and remove the ether by evaporation under reduced pressure to obtain phase A3;

[0085] S302: 10 parts of polyglyceryl 10 stearate, 5 parts of laureth-23, 1 part of a mixture of palmityl alcohol and PEG-75 stearate, glyceryl stearate and steareth-20, 10 parts of polyglyceryl-3 diisostearate, and 5 parts of hydrogenated olive oil are heated to 80° C. and mixed, and added into 100 parts of deionized water, and stirred and mixed to obtain phase B3;

[0086] S303: Add phase A3 to phase B3, mix, homogenize and shear, with a shear speed of 10,000 rpm and a shear time of 10 min. High-pressure homogenization is performed at a pressure of 100 MPa and a temperature of 80°C for 3 cycles. A pH adjuster is added to adjust the pH value to 6.5-7, and the mixture is cooled to room temperature to obtain A3-royal jelly acid nanoliposomes with antioxidant and anti-inflammatory effects.

[0087] At the same time, in order to verify the comprehensive performance of the royal jelly acid nanoliposomes prepared in the above examples, the present application provides the following comparative examples for detailed description.

[0088] Comparative Example 1

[0089] The component ratio, preparation operation and process parameters of the B1-liposome prepared in this comparative example are substantially the same as those in Example 3, except that in this comparative example, only 8.8 parts of royal jelly acid are added to prepare the B1-liposome.

[0090] Comparative Example 2

[0091] The component ratio, preparation operation and process parameters of the B2-liposome prepared in this comparative example are substantially the same as those in Example 3, except that 8 parts of royal jelly acid and 2 parts of EGCG are added in this comparative example to prepare the B2-liposome.

[0092] Comparative Example 3

[0093] The component ratio, preparation operation and process parameters of the B3-liposome prepared in this comparative example are substantially the same as those in Example 3, except that 8 parts of royal jelly acid and 0.1 parts of EGCG are added in this comparative example to prepare the B3-liposome.

[0094] Comparative Example 4

[0095] The component ratio, preparation operation and process parameters of the B4-liposome prepared in this comparative example are substantially the same as those in Example 3, except that only 8.8 parts of EGCG are added in this comparative example to prepare the B4-liposome.

[0096] The addition amount of each raw material in the examples and comparative examples of the present application is shown in Table 1:

[0097] Table 1 The amount of each raw material added in the examples and comparative examples

[0098]

[0099]

[0100] In order to verify the comprehensive performance of the liposomes prepared in the examples of the present application, corresponding performance tests were performed. The liposomes prepared in the above examples 1-3 and comparative examples 1-4 were subjected to performance tests:

[0101] Test 1: Test average particle size, Zeta potential, PDI and encapsulation efficiency

[0102] The hydrodynamic particle size, Zeta potential and polydispersity index (PDI) of the liposomes were determined by dynamic light scattering technology. The liposomes prepared in Examples 1-3 and Comparative Examples 1-4 were used as samples. After being diluted to a certain concentration with deionized water, the samples were measured three times in parallel at 25°C, and the average particle size, Zeta potential and PDI of the samples were recorded. The encapsulation efficiency was determined by high performance liquid chromatography (HPLC), and the encapsulation efficiency was calculated after liquid phase analysis. The results are shown in Table 2.

[0103] Table 2 Test results of average particle size, Zeta potential, PDI and encapsulation efficiency

[0104] Group Particle size / nm PDI Zeta potential / mV Encapsulation rate / % Example 1 156.2 0.09 -30.7 89.3 Example 2 153.4 0.11 -28.4 81.4 Example 3 153.2 0.10 -31.2 85.6 Comparative Example 1 154.6 0.13 -30.5 80.3 Comparative Example 2 153.9 0.12 -28.7 87.4 Comparative Example 3 155.4 0.12 -29.4 79.9 Comparative Example 4 155.1 0.12 -31.1 86.5

[0105] As shown in Table 2, the average particle size, Zeta potential, polydispersity index and encapsulation efficiency of the liposomes prepared in Examples 1 to 3 and Comparative Examples 1 to 4 were measured from a physicochemical perspective. It can be seen that the liposomes have uniform particle size, high encapsulation efficiency and good stability.

[0106] Test 2: Stability test

[0107] The liposomes prepared in the above Examples 1-3 and Comparative Examples 1-4 were placed in a sealed container and placed at -18°C, 4°C, room temperature and 45°C for 180 days. After returning to room temperature, the samples were checked for agglomeration or stratification at the initial state, 90 days and 180 days respectively. The results can be expressed as "yes / no" and the average particle size of the samples was tested. The results are shown in Table 3.

[0108] Table 3 Stability test results

[0109]

[0110]

[0111] The ability of cosmetics to maintain their initial properties or properties without changing over time under specified or reasonably foreseeable storage and use conditions. As shown in Table 3, the liposomes prepared in Examples 1-3 and Comparative Examples 1-4 all showed good stability, and no agglomeration and stratification occurred after being placed at -18°C, 4°C, room temperature and 45°C for 180 days. It can be seen that the preparation method provided by the present invention is of great significance for promoting the stability of the product. The particle size and particle size variation of liposome I prepared in Example 3 are the smallest, and the system is the most stable.

[0112] Test 3: ABTS free radical scavenging experiment

[0113] The liposomes prepared in the above Examples 1-3 and Comparative Examples 1-4 were used as samples, 10 μl of the sample was mixed with 190 μl of 0.25 mmol / LABTS working solution / PBS, shaken thoroughly, incubated at room temperature for 6 minutes, and the absorbance was measured at 405 nm. The absorbance of the sample and ABTS working solution was recorded as A1, the absorbance of the sample and PBS was recorded as A2, and the absorbance of the ABTS working solution and PBS was recorded as A3. Then, the ABTS clearance rate = [1-(A1-A2) / A3]×100%. The positive control was vitamin C with a loading concentration of 200, 150, 125, 100, 75, 50, and 25 μg / mL. The IC of the positive control VC was50 The test system is considered effective if the concentration is between 40 μg / mL and 70 μg / mL. Figure 1 , 2 shown.

[0114] Table 4 Test results of ABTS free radical scavenging

[0115]

[0116] ABTS is a free radical that can be reduced by antioxidants during oxidation. Figure 1 The IC of the positive control VC is 50 The test system is considered to be effective within the range of 40 μg / mL-70 μg / mL. Figure 2 It can be seen that the highest average ABTS free radical scavenging rate of the liposome emulsion prepared in Example 2 is 69.04%, and the ABTS free radical scavenging rates of Examples 1, 2, and 3 are basically consistent, all higher than the comparative example. The royal jelly acid nanoliposomes prepared in Examples 1, 2, and 3 of the present application can scavenge ABTS free radicals, and Example 3 has the best ABTS free radical scavenging performance and the best antioxidant activity.

[0117] Test 4: Cell anti-inflammatory experiment

[0118] RAW264.7 cells in the logarithmic growth phase were inoculated in a 24-well plate at a cell density of (2-5)×10 5 / mL, 0.5mL per well, incubate for 24h. After 24h, remove the supernatant and add 0.5mL of the following samples respectively:

[0119] ①Solvent control group: DMEM medium (2% FBS; 1% double antibody) was used to act on cells for 24 hours;

[0120] ②LPS inflammation model group: 1 μg / mL LPS continuously stimulated RAW264.7 cells for 24 h;

[0121] ③ Test group: Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4 + LPS (1 μg / ml) were co-acted on the cells for 24 hours.

[0122] After the sample was treated with cells for 24 hours, the supernatant was aspirated into a centrifuge tube with a pipette, centrifuged at 1000r / min for 5 minutes, the precipitate was discarded and the supernatant was aspirated, and the changes in the inflammatory factors in the supernatant were detected using TNF-α and IL-6 inflammatory factor detection kits. The detection method was strictly in accordance with the instructions on the detection kit. The results are shown in Figure 3 .

[0123] according to Figure 3It can be seen that the levels of TNF-α and IL-6 in the LPS group were significantly higher than those in the normal group, indicating that the inflammatory induction model was successfully established. Both Examples 1-3 and Comparative Examples 1-4 can cause a significant decrease in the level of inflammatory factor TNF-α in RAW 264.7 cells, and the difference is statistically significant (P < 0.0001), and the effect of Examples 1-3 in inhibiting the inflammatory factor TNF-α is better than that of the comparative example. It can be seen that the products prepared by Examples 1-3 can significantly reduce the content of inflammatory factors in cells, and the effects are stronger than those of Comparative Examples 1-4, among which the product prepared by Example 2 has the strongest effect in inhibiting inflammatory factors.

[0124] Test 5: Cytotoxicity assay

[0125] The samples of Examples 1-3 and Comparative Examples 1-4 were diluted with DMEM complete medium at a final sample concentration of 1%, and an EGCG aqueous solution with the same EGCG concentration as that in Example 1 was set as a positive control to investigate the improvement of EGCG cytotoxicity by liposome encapsulation. HDF cells (human skin fibroblasts) in logarithmic growth phase were cultured at 8×10 3 Each well was inoculated in a 96-well plate, with a system of 100 μL in each well. The outermost circle of the 96-well plate was sealed with 100 μL of PBS. After 24 hours, when the cells were completely attached to the wall, the culture medium containing the sample to be tested was replaced with a complete culture medium, and a blank group containing only culture medium and a group without EGCG package were set up. After continuing to culture the cells for 48 hours, the cell activity was detected by CCK-8 method, and the detection wavelength was 450 nm. The results are shown in Figure 4 .

[0126] Depend on Figure 4 As a result, the cell viability of the blank group was 100%, indicating that the HDF cells grew normally. Compared with the blank group, the liposome emulsions prepared by Examples 1-3 and Comparative Examples 1-4 were close to the cell viability of the blank group, and the cell viability of Comparative Example 4 decreased, but was significantly higher than that of the blank control group. The results show that Examples 1-3 and Comparative Examples 1-4 had no inhibitory effect on cells, which was close to the blank group, indicating that the liposomes provided in this application can significantly reduce the cytotoxicity of EGCG.

[0127] Test 6: Melanoma cell inhibition experiment

[0128] Determination of tyrosinase inhibition rate: Add 80μL PBS buffer, 40μL sample diluent of different concentrations, 40μL tyrosinase solution and 80μL substrate L-tyrosine solution to a 96-well plate, mix well and incubate at 25°C in the dark for 20 minutes. Then detect the absorbance at 475nm on a microplate reader, calculate the effect of the sample on the activity of elastase and calculate the statistical difference P value. The absorbance of the negative and reaction groups is recorded as A0, the absorbance of the negative blank group is recorded as A1, the absorbance of the sample and reaction group is recorded as B0, and the absorbance of the sample and blank group is recorded as B1.

[0129] Tyrosinase inhibition rate = [1-(B0-B1) / (A0-A1)] × 100%.

[0130] The positive control sample substrate is used as the sample solution for reaction control, and the negative control group, i.e., the sample diluent, is used as the sample solution for reaction control.

[0131] Table 5 Tyrosinase inhibition test results

[0132]

[0133] Note: The results of inhibition rate / clearance rate are rounded to two decimal places. The positive control concentration in the above table is 80 μg / mL. When p<0.05, it is significant. The experimental data are expressed as mean ± standard deviation, and the graph is drawn using GraphpadPrism 8 software ( Figure 5 ) and data processing, AVONA comparative analysis was used between different treatment groups.

[0134] Tyrosinase affects the synthesis of melanin, catalyzing the hydroxylation of L-tyrosine into dopa and oxidizing dopa into dopaquinone, which then forms the final reaction product, melanin. The test substance with tyrosinase activity inhibition can slow down the tyrosinase-catalyzed conversion of L-tyrosine into dopaquinone. The inhibitory effect of the test substance on tyrosinase activity is evaluated by measuring the absorbance of dopaquinone at 475 nm and the change in absorbance. According to Table 5 and Figure 5 The test results show that Examples 1-3 and Comparative Examples 1-4 can significantly inhibit tyrosinase, and the difference is statistically significant (P < 0.01). The effects of different samples show certain differences. The inhibitory effect of Examples 1-3 on tyrosinase is significantly better than that of Comparative Examples 1-4, indicating that the liposome provided in Example 3 of the present application has the best inhibitory effect on tyrosinase.

[0135] Test 7: Human patch test

[0136] This test provides a preparation of a multi-functional essence. By mass, 1 sample and 0.05 parts of sodium hyaluronate, 0.05 parts of ammonium acryloyldimethyltaurate / behenyl alcohol polyether-25 methacrylate cross-linked polymer (HMB), 0.10 parts of sodium polyacryloyldimethyltaurate (Silk), 0.10 parts of ammonium acryloyldimethyltaurate / VP copolymer (AVC), 0.5 parts of water-soluble silicone oil, 0.5 parts of 1,2-hexanediol, 0.5 parts of p-hydroxyacetophenone, and deionized water are added to 100 parts and mixed to obtain a multi-functional essence. 30 subjects were selected in the experiment and the subjects' reactions were recorded. The blank essence, the comparative example component essence, and the embodiment component essence were all attached to the curved side of the subject's forearm, and the palm of the hand was gently pressed to evenly apply it to the skin for 24 hours. After removing the spot tester, the interval was 30 minutes, and the skin reaction was observed after the indentation disappeared. The skin reaction was observed again 48 hours after the spot tester was removed. The results are shown in Table 6.

[0137] The classification of adverse skin reactions is determined according to the classification standards for adverse skin reactions in the "Safety Evaluation of Human Trial Tests" stipulated in the 2015 edition of the "Technical Specifications for Safety of Cosmetics" issued by the Ministry of Health:

[0138] Grade 0 (no skin reaction);

[0139] Grade 1 (light red spots on the skin);

[0140] Grade 2 (erythema, infiltration and papules on the skin);

[0141] Grade 3 (erythema, edema, papules and blisters on the skin);

[0142] Grade 4 (skin erythema, edema and bullae).

[0143] Table 6 Human patch test results

[0144]

[0145] The principle of the patch test is to reproduce the reaction process of allergic contact dermatitis on the local skin, that is, to observe whether a small amount of allergen directly contacts the skin and induce mild dermatitis locally, so as to judge whether the skin is allergic to the tested allergen. Figure 6 The results showed that Examples 1-3 and Comparative Examples 1-3 did not cause adverse skin reactions, while Comparative Example 4 was slightly irritating, indicating that excessive addition of EGCG would cause adverse skin reactions.

[0146] Test 8: Human efficacy test

[0147] In this test, the royal jelly acid nanoliposomes prepared in Examples 1-3 and Comparative Examples 1-4 were used as samples to prepare multi-functional essences, and human efficacy tests were performed.

[0148] Subjects: 50 people in total, 10 people in each group, numbered 1-10. The subjects used the product on their face twice a day, 1 mL each time, evenly applied on the face, for 4 consecutive weeks. The subjects were healthy volunteers with no history of cosmetic allergies.

[0149] The Visia skin detector was used to detect the improvement effect of the subjects' sensitivity (hemoglobin area / %), and the calculation method is as follows:

[0150] Total heme area reduction rate = total heme area (front) - total heme area (back) / total heme area (front) * 100%;

[0151] The test results are shown in Table 7:

[0152] Table 7 Hemoglobin test results

[0153]

[0154] According to the test results, hemoglobin can reflect the dilation of capillaries in the superficial dermis. Abnormally increased hemoglobin and capillary dilation can be used to measure the severity of skin inflammation and skin sensitivity. That is, reduced hemoglobin can support the soothing effect to a certain extent. According to Table 7 and Figure 5 The test results show that both Examples 1-3 and Comparative Examples 1-4 can reduce the total hemoglobin area and improve the skin sensitivity of the subjects. Among them, Example 3 has the best effect and the highest total hemoglobin area reduction rate.

[0155] For the same subjects, the Visia skin detector was used to characterize the parameters of human skin color through the ITA value. The calculation method is as follows:

[0156] ITA value change rate = ITA value (before) - ITA value (after) / ITA value (before) * 100%;

[0157] The test results are shown in Table 8:

[0158] Table 8 Skin color test results

[0159]

[0160] After 28 days of use, the face of the subject was tested by Visia. As shown in Table 8, compared with Comparative Examples 1-4, the light ITA values ​​of the whitening essences prepared from the liposome compositions containing royal jelly acid and EGCG in Examples 1-3 were significantly improved. Among them, the ITA value change rate of the whitening essence prepared from the liposome composition containing royal jelly acid and EGCG in Example 3 was 36.73%, which was the best effect. This shows that the whitening essence prepared from the liposome composition containing royal jelly acid and EGCG provided in the present application has a whitening effect.

[0161] The various embodiments in this specification are described in a progressive manner, and the same or similar parts between the various embodiments can be referenced to each other, and each embodiment focuses on the differences from other embodiments.

[0162] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit the present application. Although the present application has been described in detail with reference to the aforementioned embodiments, a person of ordinary skill in the art should understand that the technical solutions recorded in the aforementioned embodiments may still be modified, or some or all of the technical features therein may be replaced by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the present application.

Claims

1. A royal jelly acid nanoliposome having both anti-oxidation and anti-inflammatory effects, characterized in that: The components thereof include, by weight of raw materials: 5-10 parts of lipids, Emulsifier 2-16 parts, 1-10 parts of auxiliary emulsifier, Stabilizer 1-5 parts, 5-10 parts of royal jelly acid, Epigallocatechin gallate 0.5-1 part, 2-10 parts water.

2. The royal jelly acid nanoliposomes having both antioxidant and anti-inflammatory effects according to claim 1, characterized in that: Its components include, by weight of raw materials: 5-8 parts of lipids, Emulsifier 2-10 parts, 1-8 parts of auxiliary emulsifier, 1-3 parts of stabilizer, 5-8 parts of royal jelly acid, Epigallocatechin gallate 0.5-0.8 parts, 3-8 parts water.

3. The royal jelly acid nanoliposomes having both anti-oxidation and anti-inflammatory effects according to claim 1, characterized in that: The lipid is one or more of lecithin, caprylic / capric triglyceride, monostearate glyceryl, butyloctanol salicylate, and acetylated monoglyceride; And / or, the emulsifier is one or more of polyglyceryl 10 stearate, PEG-8 caprylic / capric glyceride, sucrose stearate, cetearyl olivate, sorbitan olivate, laureth-23, PEG-4 olivate, steareth-21, palmityl alcohol, PEG-75 stearate, glyceryl stearate, steareth-20, cetyl alcohol, cetyl alcohol ether-20, and steareth-20.

4. The royal jelly acid nanoliposomes having both anti-oxidation and anti-inflammatory effects according to claim 1, characterized in that: The auxiliary emulsifier is one or more of glycerol, 1,3-propylene glycol, 1,2-pentanediol ethoxydiglycol, olive oil PEG-7 esters, polyglyceryl-3 diisostearate, polyglyceryl-6 isostearate, polyethylene glycol-8, polyethylene glycol-12, polyethylene glycol-40, and polyethylene glycol-400.

5. The royal jelly acid nanoliposomes having both antioxidant and anti-inflammatory effects according to claim 1, characterized in that: The stabilizer is one or more of 1,3-butylene glycol, hydrogenated olive oil, olive oil, unsaponifiable matter of olive oil, cetyl palmitate, sorbitan palmitate, and sorbitan olivate.

6. The royal jelly acid nanoliposomes having both anti-oxidation and anti-inflammatory effects according to claim 1, characterized in that: The average particle size distribution of the royal jelly acid nanoliposomes with anti-oxidation and anti-inflammatory effects is 100-200 nm.

7. A method for preparing royal jelly acid nanoliposomes with anti-oxidation and anti-inflammatory effects according to any one of claims 1 to 6, characterized in that: The method comprises: The lipid, royal jelly acid and epigallocatechin gallate are dissolved in a solvent and evaporated under reduced pressure to obtain phase A; The emulsifier, co-emulsifier and stabilizer are mixed and added into water to obtain phase B; Phase A and phase B are mixed, stirred evenly, subjected to high-pressure homogenization treatment, and the pH value is adjusted to obtain the royal jelly acid nanoliposomes having both antioxidant and anti-inflammatory effects.

8. The method for preparing royal jelly acid nanoliposomes having both anti-oxidation and anti-inflammatory effects according to claim 7, characterized in that: The conditions of the high-pressure homogenization treatment are: pressure of 20-100 MPa, temperature of 40-80° C., and number of cycles of 1-5 times.

9. The method for preparing royal jelly acid nanoliposomes having both anti-oxidation and anti-inflammatory effects according to claim 7, characterized in that: The adjusted pH value is 6.5-7.

0.

10. Use of royal jelly acid nanoliposomes with antioxidant and anti-inflammatory effects prepared according to the preparation method according to any one of claims 7 to 9 in the preparation of skin care products.

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