Multi-layer sustained-release microsphere with anti-wrinkle effect as well as preparation method and application of multi-layer sustained-release microsphere

Abstract

The invention discloses a multi-layer sustained-release microsphere with an anti-wrinkle effect as well as a preparation method and application of the multi-layer sustained-release microsphere. The multilayer sustained release microsphere provided by the invention is prepared from the following components in percentage by weight: 5 to 15 percent of alligator oil, 65 to 80 percent of polylactic acid-glycolic acid copolymer (PLGA), 5 to 15 percent of chitosan and 0.1 to 5 percent of natural antioxidant. The multi-layer sustained release microsphere sequentially comprises an alligator oil-PLGA core sphere, a natural antioxidant-PLGA middle layer and a chitosan outer layer from inside to outside. The multilayer sustained-release microsphere prepared by adopting an electrostatic spinning technology has a porous multilayer structure, can realize accurate sustained release of different active components in each layer, prolongs the duration of a repairing effect, effectively reduces a burst release effect, and can ensure the stability of each active substance through layering treatment among the active substances, so that the stability of the active substances is improved. All the layers are released according to a specific rate and synergistically act on the skin, so that the utilization rate of active ingredients can be increased, waste is reduced, and meanwhile, the irritation risk to the skin is also reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and more specifically, relates to a multilayer sustained-release microsphere with anti-wrinkle effect, and a preparation method and application thereof. Background Art

[0002] As people's living standards improve, the demand for skin care products is also increasing. Wrinkles are one of the important manifestations of skin aging, and finding effective anti-wrinkle ingredients and preparation methods has become a hot topic in skin care product research. Crocodile oil is rich in unsaturated fatty acids and other nutrients, which help stimulate the production of collagen, increase skin elasticity, reduce wrinkles and sagging, delay skin aging, and have potential anti-wrinkle effects. At the same time, the use of existing crocodile oil products also has certain problems. Crocodile oil is prone to oxidation, and the oxidized crocodile oil will lose its original efficacy. In addition, under high temperature, light and other environments, crocodile oil may also stratify and even produce substances that are harmful to the skin.

[0003] In order to avoid oxidation of crocodile oil, antioxidants can be added to delay oxidation. Natural antioxidants can neutralize free radicals in the skin. Free radicals are an important cause of skin aging, and they damage DNA, lipids and proteins in skin cells. For example, vitamin E provides electrons to free radicals, preventing free radicals from further damaging skin cells, thereby delaying skin aging and reducing the formation of wrinkles. It can enhance the barrier function of the skin, make the connection between skin cells tighter, and reduce transepidermal water loss. This helps keep the skin soft and moisturized, and improves dry and rough skin conditions. While vitamin E has significant efficacy, it also has two major problems that limit its widespread application: first, poor stability. The presence of three conjugated double bonds and hydroxyl or ester groups in vitamin E makes it chemically active. Under the action of light, oxygen, heat and other factors, it is very easy to undergo oxidation, dehydration and polymerization reactions, which leads to changes in its structure, resulting in reduced retention rate and bioavailability. It is critical to protect it from oxidation and the effects of other active ingredients that lead to reduced efficacy. Second, it is highly irritating. In the early stage of use, peeling, redness and stinging may occur, and the severity varies from person to person. Therefore, it is particularly important to control the release rate of vitamin E. Therefore, there are also certain problems when using natural antioxidants.

[0004] In recent years, microsphere technology has been used to prepare a variety of drugs using microspheres as sustained-release carriers. By physically encapsulating or adsorbing drugs on the surface or inside of polymers, the release environment of drugs can be improved, their stability can be adjusted, and the sustained-release effect of drugs can be achieved. Sustained-release microspheres have shown good application prospects in the biomedical field and have become one of the hot spots in the research of sustained-release dosage forms. At present, there are single-layer microspheres loaded with drugs on the market, but the drug release control of single-layer microspheres is limited, the internal drugs are easily oxidized and easily decomposed by ultraviolet rays, the burst effect is difficult to avoid, the function is relatively single, the stability may be insufficient, and the active substances are prone to react. For example, poly(lactic-co-glycolicacid) (PLGA) is currently used to prepare microspheres, which have good biocompatibility, non-toxicity, good capsule and film-forming properties; because its degradation degree varies with the monomer ratio, it is widely used in sustained-release preparations. However, microsphere preparations with PLGA as a carrier currently also have some disadvantages, including low drug loading, high initial burst release, and poor sustained-release effect. At present, there are few studies on the application of microsphere technology in crocodile oil, and there are no crocodile oil microsphere products with sustained-release effects.

[0005] In order to improve the sustained-release effect of existing microspheres, reduce burst release, and better carry crocodile oil and natural antioxidants, while preventing the oxidation of crocodile oil, it is necessary to develop a crocodile oil sustained-release microsphere system with better sustained-release effect, good stability, not easy to oxidize and decompose, and no irritation, so as to further enhance the effect of crocodile oil, which is of great significance to the preparation and application of the sustained-release delivery system of crocodile oil. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the shortcomings of existing crocodile oil sustained-release microspheres and provide a multilayer sustained-release microsphere with anti-wrinkle effect and a preparation method and application thereof.

[0007] The object of the present invention is to provide a multilayer sustained-release microsphere.

[0008] Another object of the present invention is to provide a method for preparing multilayer sustained-release microspheres.

[0009] Another object of the present invention is to provide applications of multilayer sustained-release microspheres.

[0010] Another object of the present invention is to provide a product.

[0011] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0012] The invention provides a multilayer sustained-release microsphere, which comprises the following components in weight percentage: 5%-15% of crocodile oil, 65%-80% of polylactic acid-glycolic acid copolymer (PLGA), 5%-15% of chitosan, and 0.1%-5% of a natural antioxidant; the structure of the multilayer sustained-release microsphere is, from inside to outside, a crocodile oil-PLGA core ball layer, a natural antioxidant-PLGA middle layer, and a chitosan outer layer.

[0013] The present invention prepares crocodile oil, natural antioxidant, polylactic acid-glycolic acid copolymer, and chitosan according to a specific ratio and structure by electrospinning technology, and obtains porous multilayer sustained-release microspheres which are coated with a natural antioxidant-PLGA middle layer and a chitosan outer layer in sequence from the inside to the outside, outside the crocodile oil-PLGA core ball layer, and the particle size of the microsphere is 5μm-500μm. The sustained-release microspheres are crocodile oil as the core, and after being coated with two layers of specific layered structures, the stability of crocodile oil is improved, and a good sustained-release effect is achieved; this microsphere structure can better protect the active ingredients, and each layer can achieve accurate sustained release of different active ingredients. The crocodile oil in the core ball can be continuously released for a long time, providing deep moisturizing and repair for the skin; the natural antioxidant in the middle layer can be released at a specific rate, and while preventing the crocodile oil from being oxidized, it can also play its role in promoting skin metabolism; the chitosan in the outer layer can prevent the premature release of the ingredients and prevent the internal activity from being affected by the external environmental factors. The multilayer sustained-release microspheres provided by the present invention have the characteristics of precise sustained release, can prolong the duration of the repair effect of crocodile oil, and effectively reduce the burst effect. At the same time, the layered treatment between the active substances can ensure the stability of each active substance. Each layer is released at a specific rate and acts synergistically on the skin, which can improve the utilization rate of the active ingredients and reduce waste, and also reduce the risk of skin irritation.

[0014] Preferably, the sustained-release microspheres include the following components in weight percentage: 4%-15% crocodile oil, 70%-80% polylactic acid-glycolic acid copolymer PLGA, 4%-15% chitosan, and 0.2%-5% natural antioxidant.

[0015] Furthermore, the mass ratio of crocodile oil to PLGA in the core ball layer is 1:5-10; the mass ratio of natural antioxidant to PLGA in the middle layer is 1:10-20; the mass ratio of chitosan in the outer layer to the core ball and the PLGA in the middle layer is 1:3-6.

[0016] More preferably, the natural antioxidant is selected from one of vitamin A, vitamin C and vitamin E.

[0017] More preferably, the natural antioxidant is vitamin E.

[0018] The present invention provides a method for preparing multi-layer sustained-release microspheres, wherein a core ball layer, an intermediate layer, and a chitosan outer layer are sequentially sprayed from the inside to the outside through an electrostatic spinning technology to obtain multi-layer sustained-release microspheres. The present invention first prepares a PLGA spinning solution, adds crocodile oil to the PLGA spinning solution, and obtains a core ball solution wrapped with crocodile oil; adds a natural oxidant to the PLGA spinning solution to obtain an intermediate layer solution; and uses a chitosan solution as an outer layer solution. The solution is respectively loaded into injection pumps 1, 2, and 3, and injection pumps 1, 2, and 3 are turned on in sequence. The multi-layer sustained-release microspheres are collected on a collecting device, and the multi-layer sustained-release microspheres are obtained after being dried and washed.

[0019] The specific steps include:

[0020] S1, dissolving PLGA in an organic solvent to obtain a PLGA solution with a concentration of 10%-15%;

[0021] S2, adding crocodile oil to the PLGA solution, and then adding a porogen to obtain a core ball solution; the mass ratio of the porogen to PLGA is 1:5-10;

[0022] S3, dissolving the natural antioxidant in an organic solvent, then adding dropwise to the PLGA solution, and adding a porogen to obtain an intermediate layer solution; the mass ratio of the porogen to the PLGA is 1:5-10;

[0023] S4, dissolving chitosan in a dilute acetic acid solution to obtain a chitosan solution with a concentration of 2%-3%; then adding a porogen to obtain an outer layer solution; the mass ratio of the porogen to the chitosan is 1:5-10;

[0024] S5. Through the electrospinning technology, the core ball solution is ejected from the spinneret under the action of a high voltage electric field, and then the intermediate layer solution is sprayed to coat the outer layer of the core ball. Finally, the chitosan solution is sprayed to form a chitosan layer on the outer layer. The obtained microspheres are dried and the organic solvent is removed. Then, they are soaked, washed, and the porogen is removed to obtain multi-layered sustained-release microspheres.

[0025] Preferably, a non-polar or weakly polar organic solvent, such as isopropanol, is used in step S1.

[0026] Preferably, the porogen is sodium chloride.

[0027] Preferably, in step S6, the flow rate of the core ball solution is 0.2-0.5 mL / h, the flow rate of the middle layer solution is 0.1-0.4 mL / h, and the flow rate of the outer layer chitosan solution is 0.1-0.3 mL / h.

[0028] Preferably, the voltage of the high-voltage power supply for electrospinning is set to be 15-25 kV; the jetting distance is 10-15 cm; and the inner diameter of the spinneret is 104-130 μm.

[0029] The present invention provides application of multilayer sustained-release microspheres in preparing anti-oxidation, anti-sugar, anti-inflammatory, anti-wrinkle and anti-aging products.

[0030] The present invention provides a product comprising the multi-layer sustained-release microspheres.

[0031] Furthermore, the product is a skin care product or a cosmetic, which is prepared by a conventional preparation method in the art, specifically a lotion, an aqueous solution or a cream product.

[0032] Preferably, the product further contains moisturizers, preservatives, emulsifiers, oils and / or water.

[0033] More preferably, the product comprises the following components in weight percentage: 0.1%-10% of multilayer sustained-release microspheres, 3%-8% of moisturizer, 0.1%-0.3% of preservative, 2%-5% of emulsifier, 6%-21% of oil and fat, and the balance of water.

[0034] Further preferably, the product comprises the following components in weight percentage: 0.1%-10% of multilayer sustained-release microspheres with anti-wrinkle efficacy, 6% of moisturizer, 0.2% of preservative, 3% of emulsifier, 16% of oil and fat, and the balance of water.

[0035] It should be noted that the moisturizer, preservative, emulsifier, and oil in the multi-layer sustained-release microsphere cosmetics with anti-wrinkle efficacy of the present invention can be conventionally selected in the field, and the product prepared after compounding with the multi-layer sustained-release microspheres provided by the present invention has better efficacy; the moisturizer used in the present invention is glycerol, the preservative is potassium sorbate, the emulsifier is glyceryl monostearate, and the oil is jojoba oil and shea butter.

[0036] The present invention has the following beneficial effects:

[0037] The present invention prepares crocodile oil, natural antioxidants, polylactic acid-glycolic acid copolymer, and chitosan according to a specific weight percentage by electrospinning technology to obtain porous multilayer composite microspheres with a particle size of 5μm-500μm. This microsphere structure can better protect the active ingredients, and each layer can achieve accurate sustained release of different active ingredients. The crocodile oil in the core ball can be continuously released for a long time, providing deep moisturizing and repair for the skin; the natural antioxidant in the middle layer can be released at a specific rate to play its role in promoting skin metabolism. The multilayer sustained-release microspheres provided by the present invention have the characteristics of accurate sustained release, can prolong the duration of the repair effect, effectively reduce the burst effect, and the layered treatment between the active substances can ensure the stability of each active substance. Each layer is released at a specific rate and acts synergistically on the skin, which can improve the utilization rate of the active ingredients, reduce waste, and also reduce the risk of irritation to the skin.

[0038] At the same time, the multilayer sustained-release microspheres provided by the present invention have excellent antioxidant, anti-sugar and anti-inflammatory effects. They are prepared into anti-aging and anti-wrinkle products for the preparation of skin care cosmetics. Their ingredients are mild, safe, non-toxic, non-irritating and non-sensitizing, suitable for long-term use, and have broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the particle size distribution diagram of the microsphere sample.

[0040] Figure 2 This is a scanning electron microscope image of the microsphere sample.

[0041] Figure 3 This is a graph showing the sustained release performance of microspheres with different layers.

[0042] Figure 4 The graph shows the drug loading rate and embedding rate of microsphere samples with different numbers of layers.

[0043] Figure 5 This is the in vitro transdermal performance diagram of the microsphere samples. DETAILED DESCRIPTION

[0044] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0045] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0046] The raw materials for preparing the multilayer sustained-release microspheres used in the present invention are:

[0047] Crocodile oil is rich in unsaturated fatty acids and active ingredients. The content of unsaturated fatty acids is more than 30%. It can provide moisture and hydration for the skin, prevent dryness and roughness of the skin, and keep the skin soft and smooth. It can promote the regeneration and repair of skin cells and help improve damaged skin. The nutrients in crocodile oil can penetrate deep into the skin, provide nutrition and energy to skin cells, and promote cell metabolism and regeneration. It has a certain antioxidant effect, can resist the damage of free radicals, delay skin aging, and reduce the formation of wrinkles. It helps to increase the elasticity and firmness of the skin, making the skin younger and more energetic.

[0048] Vitamin E can capture and remove free radicals, reduce the oxidative damage of free radicals to lipids, proteins, and DNA in skin cells, and thus slow down the aging process of the skin. Skin care products containing vitamin E can promote skin metabolism, which helps to accelerate the renewal and repair of damaged skin cells. The generation of new cells can fill wrinkles and make the skin smoother.

[0049] Polylactic acid-glycolic acid copolymer (PLGA) is a polymer material with the characteristics of degradability, good biocompatibility, and non-toxicity. In the sustained-release microspheres of the present invention, polylactic acid-glycolic acid copolymer can be used as a matrix to effectively encapsulate active ingredients such as crocodile oil to form a spherical entity with a particle size distribution of 5μm-500μm. According to environmental factors such as skin temperature and pH value, the encapsulated crocodile oil can be slowly released to achieve sustained release of the drug, prolong the duration of the repair effect, and effectively reduce the burst effect, so that the drug can maintain a stable concentration for a long time and improve the therapeutic effect.

[0050] Chitosan (CS) is a natural polymer with good biocompatibility and degradability. In the sustained-release microspheres, a protective shell is provided for the microspheres to prevent the internal activity from being affected by external environmental factors and to prevent the components from being released prematurely. The porous chitosan layer is also conducive to the release of crocodile oil and vitamin E.

[0051] Example 1 Preparation of multilayer sustained-release microspheres

[0052] 1. Preparation of core ball solution, middle layer solution and outer layer solution

[0053] (1) Preparation of core ball solution: Dissolve polylactic acid-glycolic acid copolymer (PLGA80:20) in isopropanol and stir evenly to form a PLGA solution with a concentration of 10%. Then add crocodile oil to the solution, the mass ratio of crocodile oil to PLGA is 1:5, stir evenly, and make the crocodile oil evenly dispersed in the solution. Then add porogen sodium chloride to the solution to obtain a core ball solution; the mass ratio of porogen to PLGA is 1:5.

[0054] (2) Preparation of the intermediate layer solution: Weigh PLGA (80:20) and dissolve it in isopropanol to prepare a 10% PLGA solution. Dissolve the natural antioxidant vitamin E in methanol and then add it dropwise to the PLGA solution. The mass ratio of the natural antioxidant to PLGA is 1:10. Then add the porogen to the solution to obtain the intermediate layer solution. The mass ratio of the porogen to PLGA is 1:5.

[0055] (3) Preparation of outer layer solution: Dissolve chitosan in dilute acetic acid solution and stir evenly to obtain a chitosan solution of a certain concentration. Add an appropriate amount of porogen to the solution to obtain an outer layer solution; the mass ratio of porogen to chitosan is 1:5.

[0056] 2. Preparation of multilayer microspheres

[0057] Prepared by electrospinning technology, the core ball solution is loaded into injection pump 1, the middle layer solution is loaded into injection pump 2, and the outer layer chitosan solution is loaded into injection pump 3. Set the voltage of the high-voltage power supply to 15-25kV. Adjust the flow rate of the injection pump, the flow rate of the core ball solution is 0.2mL / h, the flow rate of the middle layer solution is 0.1mL / h, and the flow rate of the outer layer chitosan solution is 0.1mL / h. Set the distance between the collecting device and the spinneret to 10cm. The inner diameter of the spinneret is 104μm. Turn on the high-voltage power supply and injection pump 1, so that the core ball solution is ejected from the spinneret under the action of the high-voltage electric field, forming a fibrous structure and initially forming on the collecting device. Then turn on the injection pump 2, so that the middle layer solution is ejected, coated on the outer layer of the core ball, forming an intermediate layer containing natural antioxidants. Finally, turn on the injection pump 3, so that the chitosan solution is ejected, a chitosan layer is formed on the outer layer, and the organic solvent is dried and removed to obtain multi-layer sustained-release microspheres.

[0058] 3. Washing

[0059] The multilayer sustained-release microspheres are washed with water to dissolve and remove the porogen. The immersion time is 12-24 hours at room temperature.

[0060] Example 2

[0061] The preparation conditions of the multilayer sustained-release microspheres in this example are the same as those in Example 1, except that the mass ratio of crocodile oil to PLGA is 1:8.

[0062] Example 3

[0063] The preparation conditions of the multilayer sustained-release microspheres in this embodiment are the same as those in Example 1, except that the mass ratio of the natural antioxidant to PLGA is 1:15.

[0064] Example 4

[0065] The preparation conditions of the multilayer sustained-release microspheres in this example are the same as those in Example 3, except that a 15% PLGA solution is used.

[0066] Example 5

[0067] The preparation conditions of the multilayer sustained-release microspheres in this embodiment are the same as those in Example 1, except that the mass ratio of the porogen to PLGA and chitosan is 1:10.

[0068] Example 6

[0069] The preparation conditions of the multilayer sustained-release microspheres in this embodiment are the same as those in Example 1, with the only difference being that the distance between the collecting device and the spinneret is set to 10 cm and the inner diameter of the spinneret is 130 μm.

[0070] Example 7 Optimization of raw materials for multilayer sustained-release microspheres

[0071] 1. Selection of middle layer materials

[0072] According to the preparation method of Example 1, different intermediate layer raw materials were used for preparation, and the differences in sustained release performance and encapsulation efficiency between PLGA (polylactic acid-co-glycolic acid) and PLA (polylactic acid) as the intermediate layer materials were compared. The specific method is:

[0073] (1) Sample preparation: PLGA and PLA solutions (solvent: dichloromethane) with a concentration of 10% (w / v) were prepared respectively. Microspheres were prepared using the same emulsification-solvent evaporation method as in Example 1, with other parameters (such as stirring rate and time) kept the same, and microsphere samples were obtained after drying.

[0074] (2) Performance test: Use scanning electron microscopy (SEM) to observe the morphology of microspheres and measure the average particle size. Use HPLC to test the release of active substances in microspheres within 24 hours and draw a release curve. Calculate the encapsulation efficiency, use the direct dissolution method to extract the active substances in the microspheres and then perform quantitative analysis, and record the differences in sustained-release burst effect and encapsulation efficiency between PLGA and PLA microspheres.

[0075] The measurement results are shown in Table 1, which show that the encapsulation efficiency and burst release effect of the sustained-release microspheres prepared with PLA as the intermediate layer material are low, which is not conducive to the encapsulation and sustained release of crocodile oil, while the burst release effect and encapsulation efficiency of PLGA as the intermediate layer material are higher than those of PLA.

[0076] Table 1 Comparison of sustained release effect and encapsulation efficiency between PLGA and PLA

[0077] Material Sustained-release burst effect (24h burst rate) Encapsulation efficiency PLGA 15％ 85％ PLA 35％ 60％

[0078] 2. Selection of natural antioxidants in the middle layer

[0079] According to the preparation method of Example 1, different natural antioxidants were used to prepare the middle layer, and the effects of no antioxidant, vitamin C, or vitamin E on the stability and sustained release performance of crocodile oil were compared and evaluated. The specific method is:

[0080] (1) Sample preparation: Three groups of microsphere samples were prepared: without adding antioxidant, adding 5% (w / w) vitamin C, and adding 5% (w / w) vitamin E. The preparation method steps were consistent with the middle layer of Example 1 to ensure that the antioxidant was evenly distributed in the middle layer.

[0081] (2) Oxidation experiment: The microsphere samples were placed in an environment of 37°C and 75% relative humidity, and samples were taken after 0, 6, 12, and 24 hours. The oxidation degree of crocodile oil was tested using the peroxide value (POV) and malondialdehyde (MDA) detection methods.

[0082] (3) Performance test: HPLC was used to measure the release of crocodile oil within 24 hours to observe the effect of antioxidants on the sustained-release performance.

[0083] The test results are shown in Table 2, showing that the POV value of the vitamin E group is significantly lower than that of the vitamin C group and the group without antioxidants. Within 24 hours, the POV value of the vitamin E group was only 1.2meq / kg, while that of the vitamin C group was 2.5meq / kg and that of the group without antioxidants was 4.8meq / kg. The MDA content of the vitamin E group was 0.8nmol / mg, which was significantly lower than 1.5nmol / mg of the vitamin C group and 3.6nmol / mg of the group without antioxidants. It shows that adding natural antioxidants to the middle layer not only significantly slows down the oxidation rate of crocodile oil, but also plays a significant protective role in the stability of the active ingredients, among which vitamin E has the best antioxidant effect. Therefore, adding natural antioxidants to the middle layer in the sustained-release microspheres prepared by the present invention is a key strategy to improve the stability of crocodile oil and extend the service life of the product.

[0084] Table 2 Inhibitory effect of different antioxidants on crocodile oil oxidation (POV and MDA values)

[0085] sample POV value (meq / kg) MDA value (nmol / mg) No antioxidants 4.8 3.6 Vitamin C Group 2.5 1.5 Vitamin E group 1.2 0.8

[0086] 3. Selection of raw materials for outer layer

[0087] According to the preparation method of Example 1, different outer layer raw materials were used for preparation, and the differences in sustained release performance, free radical resistance and anti-glycosylation reaction inhibition ability between chitosan and PVA (polyvinyl alcohol) as outer layer materials were compared. The specific method is:

[0088] (1) Sustained-release performance test: Multilayer sustained-release microspheres with chitosan or PVA as the outer layer material were prepared, and the release rate of the active ingredient within 24 hours was measured.

[0089] The results are shown in Table 3, which show that chitosan exhibits a stronger sustained-release effect, especially in the first 8 hours, the release rate of the active ingredient is significantly lower than that of PVA, and can better avoid the burst release effect.

[0090] Table 3 Sustained-release effect of sustained-release microspheres with different outer layer materials

[0091] time Chitosan (EPA release rate%) PVA (EPA release rate%) 1h 16.87 28.45 4h 38.68 55.32 8h 58.86 72.45 12h 73.86 84.87 24h 88.86 95.63

[0092] (2) Anti-free radical ability: Sustained-release microspheres were prepared with chitosan or PVA as the outer layer, and their ability to scavenge DPPH free radicals was measured.

[0093] The measurement results are shown in Table 4, which show that chitosan has a stronger ability to scavenge free radicals, which is related to the porous structure formed after film formation and its stronger ability to protect active ingredients.

[0094] Table 4 DPPH free radical scavenging effect of sustained-release microspheres with different outer layer materials

[0095] Concentration (mg / mL) Chitosan(%) PVA(%) 0.2 23.56±0.86 15.98±1.05 0.6 48.32±1.89 35.87±1.45 1.0 68.26±2.43 55.76±2.01

[0096] (3) Anti-glycosylation reaction inhibition ability: Sustained-release microspheres were prepared with chitosan or PVA as the outer layer, and their inhibitory effects on non-enzymatic glycosylation reactions were measured.

[0097] The test results are shown in Table 5, which show that chitosan exhibits more significant anti-glycosylation ability, which is related to its performance of protecting active ingredients and enhancing sustained-release effect.

[0098] Table 5 Anti-glycosylation reaction inhibition effect of sustained-release microspheres with different outer layer materials

[0099] Concentration (mg / mL) Chitosan(%) PVA(%) 0.2 38.96±2.96 30.25±1.88 0.6 65.68±1.88 48.75±2.34 1.0 89.97±2.36 68.26±2.98

[0100] Based on the above effects, the differences in performance indicators and physicochemical properties between chitosan and PVA as outer layer materials are further compared. The specific comparison results are shown in Table 6, which shows that chitosan should be preferred as the outer layer material in scenarios with high requirements for sustained release and protection of active ingredients.

[0101] Table 6 Comparison of performance indicators and physical and chemical properties of outer layer materials

[0102]

[0103] Example 8 A product with anti-wrinkle effect

[0104] The present embodiment provides a product with anti-wrinkle effect, specifically comprising the above-mentioned multi-layer sustained-release microsphere cosmetic product, which also contains auxiliary ingredients commonly used in the cosmetics field, specifically comprising the following components in weight percentage: 0.1%-10% of crocodile oil multi-layer sustained-release microspheres, 3%-8% of moisturizer, 0.1%-0.3% of preservative, 2%-5% of emulsifier, 6%-21% of oil, and the balance is water.

[0105] After the crocodile oil multilayer sustained-release microspheres are prepared into cosmetics by conventional methods in the art, the multilayer sustained-release microspheres can also exert their effects and have good anti-wrinkle effects. The optimized multilayer sustained-release microsphere cosmetics contain the following weight percentage components: 0.1%-10% crocodile oil multilayer sustained-release microspheres, 6% moisturizer, 0.2% preservative, 3% emulsifier, 16% oil and the rest water; the anti-wrinkle cream prepared by the formula has the best skin feel, is easier to be absorbed by the skin, and is refreshing and not sticky.

[0106] It should be noted that the moisturizer, preservative, emulsifier, and oil used in this embodiment are conventional choices of raw materials in the field of cosmetics. Specifically, the moisturizer is glycerol, the preservative is potassium sorbate, the emulsifier is glyceryl monostearate, and the oils are jojoba oil and shea butter.

[0107] Furthermore, the multi-layer sustained-release microsphere cosmetics with anti-wrinkle effects can be prepared into products such as emulsions, aqueous solutions, essences, creams, etc. by conventional preparation methods in the art.

[0108] Comparative Example 1: Single-layer sustained-release microspheres

[0109] S1, dissolving natural antioxidant vitamin E in water to obtain a 0.5 mol / L vitamin E solution;

[0110] S2, mixing crocodile oil with the vitamin E solution in step 1 at a ratio of 3:1 at 30-50° C., and stirring for ten minutes using a high-speed stirrer (500-2000 rpm) to obtain a mixed solution;

[0111] S3, dissolving polylactic acid-co-glycolic acid (PLGA 80:20) in isopropanol and stirring evenly to form a PLGA solution with a concentration of 10%;

[0112] S4, slowly adding the mixed solution of step 2 to the polymer solution of step S3, and controlling the mass ratio of crocodile oil to polylactic acid-glycolic acid to be 1:5, and obtaining the drug-loaded colostrum under high-speed stirring (1000-5000 rpm);

[0113] S5, dissolving polyvinyl alcohol (PVA) in deionized water to prepare a polyvinyl alcohol solution (concentration of 5‰ g / mL);

[0114] S6, mixing the polyvinyl alcohol solution of step 5 with the drug-loaded colostrum solution of step 4, and then performing ultrasonic emulsification to obtain a drug-loaded emulsion;

[0115] S7, slowly dropwise adding the drug-loaded emulsion solution of step 6 into the polyvinyl alcohol dispersion (concentration of 5‰ g / mL), the volume ratio of the drug-loaded emulsion to the PVA dispersion being 1:10; stirring with magnetic force at constant temperature for 1 hour to solidify the drug-loaded PLGA microspheres;

[0116] S8. The emulsion in step 7 is subjected to ice bath extraction, ion water washing, and freeze drying to obtain single-layer sustained-release microspheres.

[0117] Comparative Example 2: Three-layer sustained-release microspheres

[0118] S1. Dissolve PLGA (80:20) in isopropanol and stir well to form a 10% PLGA solution. Then add crocodile oil to the solution, the mass ratio of crocodile oil to PLGA is 1:5, stir well to make the crocodile oil evenly dispersed in the solution. Then add sodium chloride to the solution, the mass ratio of sodium chloride to PLGA is 1:5, and obtain a core ball solution;

[0119] S2. Dissolve PLGA (80:20) in isopropanol to prepare a 10% PLGA solution. Dissolve the natural antioxidant in methanol and then add it dropwise to the PLGA solution. The mass ratio of the natural antioxidant to PLGA is 1:10. Then add sodium chloride to the solution. The mass ratio of sodium chloride to PLGA is 1:5 to obtain the first layer of solution from the inside to the outside.

[0120] S3, dissolving polyvinyl alcohol (PVA) in water, stirring evenly, then adding a porogen to the solution, stirring evenly, the mass ratio of the porogen to the polyvinyl alcohol being 1:5, and obtaining a second layer of solution from the inside to the outside;

[0121] S4. Dissolve chitosan in a dilute acetic acid solution, then add a porogen to the solution and stir evenly. The mass ratio of the porogen to chitosan is 1:5, and a third layer of solution is obtained from the inside to the outside.

[0122] S5. Load the core ball solution into injection pump 1, the first layer solution into injection pump 2, the second layer solution into injection pump 3, and the third layer solution into injection pump 4. Set the voltage of the high-voltage power supply to 15-25kV. Adjust the flow rate of the injection pump, the flow rate of the core ball solution is 0.2mL / h, the flow rate of the middle layer solution is 0.1mL / h, the flow rate of the sub-outer layer solution is 0.1mL / h, and the flow rate of the outer layer solution is 0.1mL / h. Set the distance between the collecting device and the spinneret to 10cm. The inner diameter of the spinneret is 104μm. Turn on the high-voltage power supply and injection pump 1, so that the core ball solution is ejected from the spinneret under the action of the high-voltage electric field, forming a fibrous structure and initially forming it on the collecting device. Then turn on the injection pump 2, so that the first layer of solution is ejected, coated on the outer layer of the core ball, forming an intermediate layer. Then turn on the injection pump 3, so that the second layer of solution is ejected to form a sub-outer layer. Finally, the syringe pump 4 is turned on to spray out the third layer of solution to form an outer layer, and then dried and the organic solvent is removed to obtain three-layer sustained-release microspheres;

[0123] S6, washing the microspheres in step 5 with water to dissolve and remove the porogen. The immersion time is 12-24 hours at room temperature.

[0124] Test Example 1 Particle Size Distribution of Multilayer Sustained-Release Microspheres

[0125] Take an appropriate amount of the multilayer sustained-release microsphere sample prepared in Example 1 and put it into a container filled with water and ethanol. Use an ultrasonic disperser to disperse the sample to ensure that the microspheres are evenly dispersed in the medium to avoid agglomeration. Instrument calibration: According to the instrument manual, use standard particles to calibrate the laser particle size analyzer to ensure the accuracy of the measurement. Inject the dispersed sample into the sample cell of the laser particle size analyzer and start the measurement program. The laser irradiates the sample, and the detector receives the scattered light signal. By analyzing the intensity and angular distribution of the scattered light, the particle size and distribution of the microspheres are calculated.

[0126] Test results such as Figure 1 As shown, the particle size of the microspheres is normally distributed, with an average particle size of approximately 70 μm, but overall meets the design requirements of the 5 μm-500 μm range. The microspheres within this range have the advantages of high permeability and long-acting sustained release, adapting to a variety of release needs and application scenarios, and have good processing flexibility and market potential.

[0127] Test Example 2 Surface morphology of multilayer sustained-release microspheres

[0128] A scanning electron microscope (SEM) is used to measure the surface morphology. First, the multilayer sustained-release microsphere sample prepared in Example 1 is fixed on a sample stage. It is usually dried and subjected to a conductive treatment (such as gold spraying or carbon coating) to prevent charge accumulation. Then the sample is placed in a SEM instrument, and appropriate parameters such as acceleration voltage and beam current are set. The electron beam scans the sample surface, and the generated signal is received by the detector and converted into an image. The microspheres are observed to observe the surface roughness of the microspheres and the morphology of the microspheres. The measurement results are shown in FIG. Figure 2 As shown, the microsphere particles are relatively uniform in size, with a particle size of about 100-150 μm.

[0129] Test Example 3 Sustained Release Performance of Sustained Release Microspheres

[0130] The release rate of the active ingredient in the multilayer sustained-release microsphere sample prepared in Example 1 was determined by gas chromatography. 100 mg of the multilayer sustained-release microsphere sample was accurately weighed and placed in a centrifuge tube, 10 mL of n-hexane was added to immerse it, 100 mg of the multilayer sustained-release microsphere sample was accurately weighed and placed in a sealed sample bottle or dialysis bag, 10 mL of n-hexane was added to immerse it, and the sample bottle containing the sustained-release microsphere and the release medium was placed in a 37°C constant temperature oscillator to simulate the human body environment. The sustained-release effect of crocodile oil was characterized by measuring the core components of crocodile oil, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).

[0131] Use a medium polarity capillary column DB-5MS suitable for fatty acids and vitamin E. The nitrogen flow rate is 1-3mL / min, and the injection port temperature is set to 250°C. The column temperature program starts at 50°C, maintained for 1 minute, increased to 200°C at a rate of 10°C / min, and then increased to 280°C at 5°C / min and maintained for 10 minutes to ensure that all components are separated. The flame ionization detector (FID) temperature is 250-300°C. Prepare standard solutions of EPA, DHA and vitamin E (such as 0.1, 0.5, 1, 2 mg / mL), inject and measure the peak area or peak height respectively, and draw three standard curve equations for EPA, DHA and vitamin E respectively.

[0132] Place the microsphere sample in the release medium and maintain a stable oscillation speed in a constant temperature oscillator at 37°C. Extract 1 mL of the release medium as a sample at set time points (1h, 2h, 4h, 8h, 12h, 24h) to ensure the uniformity of the release medium. Add the same volume of fresh release medium after each sampling to maintain the total volume. Inject the sample at each time point into the gas chromatograph and record the chromatographic peak area or peak height of EPA, DHA and vitamin E respectively. Calculate the concentration at each time point based on the standard curve. Through the concentration changes at different times, the sustained release curves of the three components can be drawn to analyze their sustained release characteristics. Compare the release curves of different components and analyze the release rate, sustained release time and burst release phenomenon of each component. The ideal sustained-release microsphere should show a gradual release curve to avoid initial burst release or rapid release.

[0133] Release rate = C t / C total ×100%

[0134] Among them C t is the concentration of active ingredient at a certain time point; C total is the total concentration of active ingredients.

[0135] The results are shown in Table 7 and Figure 3 As shown, the active ingredients EPA and DHA of crocodile oil were used as the main measurement indicators, and the release effect of the natural antioxidant vitamin E was measured at the same time. The results showed that the release rate of the single-layer sustained-release microspheres of Example 1 was faster and the burst release effect was obvious. The release rate of the two-layer sustained-release microspheres prepared in Example 1 was relatively stable, and sustained and effective release could be achieved. The release curve of the three-layer sustained-release of Example 2 showed that its burst release effect was significantly higher than that of the two-layer sustained-release in the initial stage, but the subsequent release gradually slowed down and failed to achieve complete release; after 24 hours, the total release rate of the three-layer sustained-release was significantly lower than that of the two-layer sustained-release, indicating that there was a problem of incomplete release of the active ingredients.

[0136] Table 7 Active ingredient release effects of different sustained-release microspheres

[0137]

[0138] Test Example 4 Test of precise sustained-release effect of each layer of multi-layer sustained-release microspheres

[0139] According to the method of Example 1, a multilayer sustained-release microsphere was prepared by adding a marking dye when preparing each layer of solution, and a multilayer sustained-release microsphere containing a specifically marked active ingredient was prepared (the crocodile oil in the core ball layer was marked with fluorescent dye A, and the natural antioxidant in the middle layer was marked with fluorescent dye B. The added dye did not affect the properties and sustained-release function of the active ingredient). A certain amount of multilayer sustained-release microspheres containing the marked active ingredient was accurately weighed and divided into three groups, each group having the same mass, and used for testing at different time points.

[0140] Take an appropriate amount of phosphate buffered saline (PBS) and simulated skin permeation fluid (the ingredients simulate the ingredients of skin tissue fluid, used to simulate the skin environment) and mix them in a certain proportion to prepare the release medium. Ensure that the composition and properties of the release medium can simulate the release conditions of the microspheres in the skin environment. The three groups of samples were placed in three centrifuge tubes containing the release medium, and the same volume of release medium was added to each centrifuge tube to ensure that the microspheres were completely immersed in the medium. The centrifuge tube was placed in a constant temperature oscillator, the temperature was set to 37°C (simulating human skin temperature), and the oscillation speed was 100rpm (according to the preliminary experiment, the appropriate oscillation speed was determined to simulate the dynamic environment of the skin). At 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours and other time points, a certain volume of release medium was taken out from each centrifuge tube (the same volume was sampled each time), and the same volume of fresh release medium was added to keep the total volume unchanged. The amount of active ingredient released in each layer was detected.

[0141] 1. Core ball layer (crocodile oil): Centrifuge the taken-out release medium to remove possible impurities such as microsphere fragments. Use a fluorescence spectrophotometer to detect the fluorescence intensity of fluorescent dye A in the release medium. According to the pre-made standard curve of fluorescence intensity and crocodile oil concentration (made by labeling samples with known concentrations of crocodile oil), calculate the concentration of crocodile oil in the release medium, and thus obtain the crocodile oil release amount of the core ball layer at different time points.

[0142] 2. Intermediate layer (natural antioxidant): The taken-out release medium is also centrifuged. The fluorescence intensity of the fluorescent dye B in the release medium is detected using a fluorescence spectrophotometer. The concentration of the natural antioxidant in the release medium is calculated based on the pre-made standard curve of fluorescence intensity and natural antioxidant concentration (made by labeling samples with known concentrations of natural antioxidants), thereby obtaining the amount of natural antioxidant released in the intermediate layer at different time points.

[0143] 3. The concentration of all active ingredients (including crocodile oil and natural antioxidants) in the taken-out release medium is measured by high performance liquid chromatography (HPLC). By comparing the total active ingredient concentration measured by HPLC with the sum of the active ingredient concentrations of each layer measured by fluorescence spectrophotometer, it is verified whether the sum of the release amounts of each layer is equal to the overall release amount, and whether the release of each layer meets the characteristics of precise sustained release.

[0144] 4. Observe the changes in the internal structure of the microspheres: At certain time points (4 hours, 12 hours, and 24 hours), take out some microsphere samples from the constant temperature oscillator. Use a laser confocal microscope to observe the changes in the internal structure of the microspheres. Fix the microspheres on a suitable glass slide, and observe the integrity of the internal structure of each layer of the microspheres and the distribution changes of the active ingredients in each layer by fluorescence imaging of the labeled active ingredients, and further verify the sustained release effect of each layer.

[0145] The results are shown in Table 8. Core ball layer (crocodile oil): In the initial stage (0.5-1 hour), the crocodile oil release is less, and gradually increases over time, but the release rate is relatively stable. The release is about 2% (percentage of the total amount of crocodile oil in the core ball layer) at 0.5 hour, about 4% at 1 hour, about 8% at 2 hours, about 15% at 4 hours, about 25% at 8 hours, about 40% at 12 hours, and about 60% at 24 hours.

[0146] Middle layer (natural antioxidant): Its release curve is different from that of the core ball layer, and it is released at its own specific rate. The release amount may be about 3% (the percentage of the total amount of natural antioxidants in the middle layer) at 0.5 hours, about 6% at 1 hour, about 10% at 2 hours, about 18% at 4 hours, about 30% at 8 hours, about 45% at 12 hours, and about 65% at 24 hours. In addition, the release rate of natural antioxidants is different from that of crocodile oil in the core ball layer, reflecting the precise slow-release characteristics of each layer at a specific rate.

[0147] Table 8 Release amount of active substances in each layer at different times

[0148]

[0149] By comparing the HPLC measurement of the overall multilayer microspheres and the fluorescence spectrophotometer measurement results of each layer, it was verified that the sum of the release amounts of each layer was consistent with the overall release amount, further proving that each layer can achieve precise sustained release, and the synergistic effect between the layers can jointly achieve the ideal sustained release effect of the multilayer sustained-release microspheres. Each layer can achieve precise sustained release of different active ingredients. The crocodile oil in the core ball can be continuously released for a long time to provide deep moisturizing and repair for the skin; the natural antioxidants in the middle layer can be released at a specific rate to play its role in promoting skin metabolism. This precise sustained-release feature can improve the utilization rate of active ingredients, reduce waste, and also reduce the risk of skin irritation.

[0150] Test Example 5 Drug loading and encapsulation efficiency of sustained-release microspheres

[0151] Select n-hexane as the solvent, prepare the standards of EPA, DHA and vitamin E, dissolve them in the solvent respectively, and prepare a series of solutions with known concentrations (e.g. 0.1, 0.5, 1, 2 mg / mL). According to the characteristic absorption peaks of EPA, DHA and vitamin E, use a UV-visible spectrophotometer to determine the optimal detection wavelength of each component. Measure the absorbance of each concentration standard solution at the optimal wavelength, and draw a standard curve of concentration (abscissa) and absorbance (ordinate) for subsequent concentration calculation.

[0152] Accurately weigh 100 mg of sustained-release microsphere sample (Example 1, Comparative Example 1-2), and record the total mass M total The microsphere sample was placed in an appropriate amount of ethanol solvent ethanol, and ultrasonic treatment or heating stirring was used to ensure that the microspheres were completely dissolved and EPA, DHA and vitamin E were released.

[0153] Take an appropriate amount of solution and use a UV-visible spectrophotometer to measure the absorbance at the characteristic wavelength of each component. Determine the concentration of each component C using the standard curve total , and calculate its mass using the formula: W drug-total =C total ×V solution , where V solution Solution volume. Drug loading = W drug-total / M total ×100%

[0154] Determination of unencapsulated components: Take a sample of sustained-release microspheres of the same mass, soak them in ethanol solvent, centrifuge the microspheres and collect the supernatant (this supernatant contains unencapsulated EPA, DHA and vitamin E). Measure the absorbance of the supernatant at the characteristic wavelength corresponding to the standard curve. The concentration of the unencapsulated components in the supernatant is obtained through the standard curve, and the mass of the unencapsulated components is calculated using the formula W drug-free :

[0155] Wdrug-free =C free ×V supernatant

[0156] Where V supernatant is the volume of supernatant;

[0157] Encapsulation efficiency = (W drug-total -W drug-free ) / M total ×100%;

[0158] The theoretical feed amount refers to the initial addition amount of EPA, DHA and vitamin E in the experimental formula.

[0159] The results are shown in Table 9 and Figure 4 As shown, the single-layer sustained-release microspheres of comparative example 1 were measured to have a drug loading of 15% and an encapsulation efficiency of 50%. The three-layer sustained-release microspheres of comparative example 3 were measured to have a drug loading of 20% and an encapsulation efficiency of 60%. The multi-layer (two-layer) sustained-release microspheres of Example 1 were measured to have a drug loading of 30% and an encapsulation efficiency of 85%, both of which were higher than the single-layer and three-layer sustained-release microspheres.

[0160] Table 9 Drug loading rate and embedding rate of microspheres with different layers

[0161]

[0162] Test Example 5 Scavenging effect of sustained-release microspheres on DPPH free radicals

[0163] To determine the DPPH free radical scavenging ability of the sustained-release microspheres, several test tubes were taken, 2 mL of 0.1 mmol 1 L DPPH solution was added to each test tube, and the test tubes were placed in an ice water bath to avoid light, and then different concentrations of sustained-release microspheres (Examples 1-6, Comparative Examples 1-2) and anhydrous ethanol were added in sequence to make the total volume of the solution in each test tube 4 mL. After 30 minutes of reaction in the dark, the absorbance of each sample at 517 nm was measured using an ultraviolet-visible spectrophotometer with anhydrous ethanol as a reference.

[0164] The results of the free radical scavenging effect are shown in Table 10 below, which show that the multilayer sustained-release microspheres prepared by the present invention have better free radical scavenging effect. When the microsphere concentration is 0.2 to 1 mg / mL, the DPPH free radical scavenging rate is 24.34% to 93.37%, and the scavenging rate increases with the increase of its concentration, and there is an obvious dose-effect relationship.

[0165] Table 10 Scavenging effect of sustained-release microspheres on free radicals

[0166]

[0167]

[0168] Test Example 6 Inhibition rate of sustained-release microspheres on non-enzymatic glycosylation reaction

[0169] 19.82g glucose, 0.4024g bovine serum albumin and 0.39g sodium azide were added to 100mL of 0.2mol / L phosphate buffer with pH=7.4, and mixed to form a reaction solution. Several test tubes were taken, and 4mL of reaction solution and sustained-release microspheres of different concentrations (Examples 1-6, Comparative Examples 1-2) were added to each test tube in sequence, and then the microspheres were dissolved and mixed with analytical grade anhydrous ethanol to form an ethanol solution with a volume fraction of 50%, so that the total volume of the solution in all test tubes was 5mL. The initial fluorescence value of all samples was measured by a fluorescence spectrophotometer, and then all test tubes were placed in a 37°C water bath in the dark for 7 days, and then the fluorescence value of the sample in each test tube was measured, and all test tubes were kept in a 37°C water bath in the dark for 7 days, and then the fluorescence value was measured.

[0170] The test conditions are: excitation wavelength 370nm, slit 5nm; emission wavelength range 380-500nm, slit 5nm; light source voltage 700V. The inhibition rate is calculated using the obtained fluorescence data, and the calculation formula is IR (inhibition rate) = 1-F (1-5) / F6, (F is the fluorescence value of different test tubes).

[0171] The results of the non-enzymatic glycosylation reaction are shown in Table 11 below. There is a close connection between non-enzymatic glycosylation and oxidation reaction. Oxidation reaction is the middle and late stages of non-enzymatic glycosylation reaction, and glycosylation reaction promotes oxidation reaction at the same time. The results show that the multilayer sustained-release microspheres prepared by the present invention have better antioxidant activity and can effectively inhibit non-enzymatic glycosylation reaction.

[0172] Table 11 Inhibitory effect of sustained-release microspheres on non-enzymatic glycosylation reaction

[0173]

[0174]

[0175] Test Example 7 Protective Effects of Sustained-Release Microspheres on Oxidative Stress and Inflammation

[0176] Oxidative stress and inflammation are the main factors that cause skin photodamage. Further research was conducted using an enzyme-linked immunosorbent assay (ELISA), taking the blank group, the model group, and the multilayer microspheres of Example 1 and setting different concentrations for treatment: 1% low concentration (L), 5% medium concentration (M), and 10% high concentration (H) for the skin tissue of mice treated, and setting the vitamin E group as a positive control (the same dosage for each treatment group). Take the treated mouse skin tissue, homogenize the skin tissue of each group and take the supernatant, and measure the levels of IL-1β, IL-6, COX-2, and TNF-α in the skin tissue homogenate. The specific operation strictly refers to the instructions of the kit.

[0177] The results are shown in Table 12 below, showing that under UVB induction, compared with the blank group, the inflammatory factors IL-6 and TNF-α in the skin of the model group increased significantly (P<0.01), and IL-1β and COX-2 also had an upward trend. It is worth noting that compared with the blank group, the high-concentration multilayer sustained-release microsphere group of Example 1 can significantly reduce IL-1β in the skin (P<0.05), significantly reduce IL-6 in the skin to (66.05±2.88) pg / mL (P<0.05), COX-2 significantly decreased to (6.38±0.22) ng / mL (P<0.05), and TNFα significantly decreased to (5.35±0.56) pg / mL (P<0.01). Compared with the model group, vitamin E can significantly reduce COX-2 and TNF-α levels (P<0.05). Compared with the vitamin E group, the IL-1β, IL-6, TNF-α, and COX-2 in the medium and low concentration microsphere groups of Example 1 all showed a downward trend.

[0178] Table 12 Effects of the test substances on inflammatory factors in UVB-induced photoaging skin of ICR mice

[0179] Group IL-1β / pg / mL IL-6 / pg / mL COX-2 / ng / mL TNF-α / pg / mL Blank group 68.09±3.68 83.99±4.67 6.99±0.34 12.20±1.41 Model Group 183.80±3.65 196.63±6.90 12.05±0.61 32.33±1.28 Vitamin E group 78.15±6.40 102.05±6.42 7.68±0.52 26.02±2.59 H 76.63±3.20 66.05±2.88 6.38±0.22 5.35±0.56 M 80.64±4.26 90.78±3.80 8.42±0.23 9.85±0.50 L 98.86±3.20 128.12±34.23 12.84±0.24 23.95±3.41

[0180] The above results show that the multilayer sustained-release microspheres prepared by the present invention can significantly reduce the levels of oxidative stress and inflammatory factors IL-1β, IL-6, COX-2, and TNF-α, and the improvement effect is positively correlated with the dosage concentration, avoiding skin photodamage and having a good protective effect on UVB radiation-induced skin damage in mice.

[0181] Test Example 8 In vitro skin permeability test

[0182] Standard solutions of EPA, DHA and vitamin E were used to prepare standard solutions of different known concentrations, and the absorbance or peak area was measured by high performance liquid chromatography (HPLC) to draw a standard curve for subsequent quantitative calculation of data.

[0183] The membrane material simulating the skin is installed between the supply tank and the receiving tank of the transdermal diffusion instrument to ensure that the membrane has good sealing. An appropriate amount of receiving liquid is added to the receiving tank, and the stirring device and the temperature control system are turned on to keep the receiving liquid at a certain temperature and stirring speed. The test solution is added to the supply tank to make the multilayer microsphere sample of Example 1 contact with the simulated skin. At a certain time interval, a certain volume of receiving liquid is taken out from the receiving tank, and the same volume of fresh receiving liquid is added to keep the volume in the receiving tank constant.

[0184] High performance liquid chromatography (HPLC) was used to analyze the concentrations of EPA, DHA and vitamin E in the receiving solution. Based on the concentration data at different time points, the transdermal rate of each component was calculated, and its sustained release characteristics and transdermal effect were analyzed.

[0185] The results are shown in Table 13 and Figure 5 As shown, the concentration of the active ingredient in the receiving fluid at different time points is: at the 1st hour, the concentration of the active ingredient in the receiving fluid is 0.05 mg / mL; at the 2nd hour, it is 0.1 mg / mL; at the 4th hour, it is 0.2 mg / mL; at the 8th hour, it is 0.35 mg / mL; at the 12th hour, it is 0.5 mg / mL; and at the 24th hour, it is 0.65 mg / mL.

[0186] Transdermal rate analysis: According to the above concentration data, the transdermal rate was calculated. The average transdermal rate in the first 2 hours was about 0.025 mg / (cm 2 ·h), the average transdermal rate in 4-8 hours is about 0.03mg / (cm 2 ·h), the average transdermal rate in 12-24 hours is about 0.02mg / (cm 2 h).

[0187] Transdermal volume analysis: within 24 hours, the total transdermal volume is about 1.85 mg / cm 2 .

[0188] Table 13 Active ingredient concentrations in the receiving solution at different time points

[0189]

[0190] In summary, the results of Table 13 show that the multilayer sustained-release microspheres have a release rate of nearly 90% within 24 hours, a low initial burst effect, and a stable and uniform release rate, showing the advantages of precise sustained release and high release efficiency, and can make full use of drug-carrying substances, reduce waste, and significantly prolong the action time, avoiding the problem of incomplete release. The multilayer sustained-release microspheres containing crocodile oil prepared by the present invention can be used in the field of cosmetics to prepare delayed-release skin care products, perform antioxidant, anti-sugar, and anti-inflammatory effects, and thus serve as anti-aging and anti-wrinkle products, such as preparing anti-wrinkle creams, repair essences, etc.; in the field of medicine, it can be used for the preparation of external or transdermal drugs such as anti-inflammatory ointments and analgesic patches, and can be expanded to tissue repair carriers in biomedicine and slow-release fertilizers or pesticides in agriculture, etc., to achieve precise sustained release and long-lasting effects.

[0191] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A multilayer sustained-release microsphere, characterized in that: The multilayer sustained-release microspheres include the following components in weight percentage: 5%-15% crocodile oil, 65%-80% polylactic acid-glycolic acid copolymer PLGA, 5%-15% chitosan, and 0.1%-5% natural antioxidant; the structure of the multilayer sustained-release microspheres from inside to outside is: crocodile oil-PLGA core ball, natural antioxidant-PLGA middle layer, and chitosan outer layer.

2. The multilayer sustained-release microsphere according to claim 1, characterized in that: The sustained-release microspheres comprise the following components in weight percentage: 4%-15% crocodile oil, 70%-80% polylactic acid-glycolic acid copolymer PLGA, 4%-15% chitosan, and 0.2%-5% natural antioxidant.

3. The multilayer sustained-release microspheres according to claim 1, characterized in that: The mass ratio of crocodile oil to PLGA in the core ball is 1:5-10; the mass ratio of natural antioxidant to PLGA in the middle layer is 1:10-20; the mass ratio of chitosan in the outer layer to the core ball and the PLGA in the middle layer is 1:3-6.

4. The multilayer sustained-release microsphere according to any one of claims 1 to 3, characterized in that: The natural antioxidant is selected from one or more of vitamin A, vitamin C and vitamin E.

5. The method for preparing the multilayer sustained-release microspheres according to any one of claims 1 to 3, characterized in that: The core ball, the middle layer and the outer layer are sprayed in sequence from the inside to the outside through the electrospinning technology to obtain multi-layer sustained-release microspheres.

6. The method according to claim 5, characterized in that: The specific steps include: S1, dissolving PLGA in an organic solvent to obtain a PLGA solution with a concentration of 10%-15%; S2, adding crocodile oil to the PLGA solution, and then adding a porogen to obtain a core ball solution; The mass ratio of porogen to PLGA is 1:5-10; S3, dissolving the natural antioxidant in an organic solvent, then adding dropwise to the PLGA solution, and adding a porogen to obtain an intermediate layer solution; the mass ratio of the porogen to the PLGA is 1:5-10; S4, preparing a chitosan solution with a concentration of 2%-3%; Then, a porogen is added to obtain an outer layer solution; the mass ratio of the porogen to chitosan is 1:5-10; S5. Through the electrospinning technology, the core ball solution is ejected from the spinneret under the action of a high voltage electric field, and then the intermediate layer solution is sprayed to coat the outer layer of the core ball. Finally, the chitosan solution is sprayed to form a chitosan layer on the outer layer. The obtained microspheres are dried and the organic solvent is removed. Then, they are soaked, washed, and the porogen is removed to obtain multi-layered sustained-release microspheres.

7. The method according to claim 6, characterized in that: In step S5, the flow rate of the core ball solution is 0.2-0.5 mL / h, the flow rate of the middle layer solution is 0.1-0.4 mL / h, and the flow rate of the outer layer chitosan solution is 0.1-0.3 mL / h.

8. Use of the multilayer sustained-release microspheres according to any one of claims 1 to 4 in the preparation of anti-oxidation, anti-sugar, anti-inflammatory, or anti-wrinkle and anti-aging products.

9. A product, characterized in that Containing the multilayer sustained-release microspheres according to any one of claims 1 to 4.

10. The product according to claim 9, characterized in that: Also contains humectants, preservatives, emulsifiers, oils and / or water.

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