Liposome of lipoic acid, and preparation method and application thereof

Abstract

The application relates to the technical field of liposome preparation, in particular to a lipoic acid liposome as well as a preparation method and application thereof. The raw materials of the lipoic acid liposome of the application include lipoic acid, a mixed solvent, mixed lipids and a stabilizer; wherein the mixed lipids include hydrogenated lecithin, camellia seed oil and tocopherol acetate. Through improvement of the raw material formula and the preparation process of the lipoic acid liposome, the product stability and the use safety are remarkably improved, the transdermal absorption performance is remarkably improved, long-acting release is realized, multiple components are loaded, and the whitening and anti-wrinkle effects of the product are remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of liposome preparation technology, and in particular to a lipoic acid liposome, its preparation method, and its application. Background Technology

[0002] Alpha-Lipoic Acid (ALA) is a powerful all-around antioxidant, often added to vitamins and anti-aging agents. However, its practical applications still have significant limitations.

[0003] The main disadvantages of lipoic acid are as follows: (1) Lipoic acid is extremely unstable and very sensitive to light, heat and oxygen. It is prone to degradation and dimerization, which leads to the rapid loss of its antioxidant activity. The related products will quickly turn yellow and become ineffective under light. During the production process (such as high temperature sterilization and emulsification) or when stored at high temperature in summer, the active ingredients will degrade in large quantities. After opening, they will come into contact with oxygen in the air and gradually deteriorate, resulting in a short shelf life for the related products; (2) High concentrations of L-lipoic acid will cause discomfort such as stinging, fever and redness on human skin. Its strong acidity and possible effects on certain skin receptors or nerve endings require higher tolerance; (3) Lipoic acid has a low transdermal absorption rate. When applied to the skin, a large amount of active ingredients remain on the skin surface and cannot penetrate into the target point (such as the dermis) to exert the corresponding effect; (4) Poor formulation compatibility. Lipoic acid is active and easily interacts with other components in the formulation (such as certain metal ions and other active agents), resulting in instability of the entire formulation system (such as discoloration, layering, and viscosity changes), which limits its compound use. Chinese patent CN101721369A discloses a method for preparing α-lipoic acid liposomes. This method involves dissolving soybean lecithin and cholesterol in an organic solvent, then adding the solution to α-lipoic acid to form a liposome solution. The mass ratio of soybean lecithin, cholesterol, and α-lipoic acid used is 3.1~3.5:1:0.03~0.06. The solution is then subjected to water bath, reduced pressure, and rotary evaporation to form a thin film. The auxiliary agent diethyl ether is added, followed by hydration and fragmentation to form homogeneous liposomes. The liposomes prepared using this method are uniform, stable, and have a high encapsulation efficiency, which is beneficial for industrial production.

[0004] Liposomes are tiny spherical carriers composed of a phospholipid bilayer. Their core advantages lie in their biomimetic structure and multifunctional drug delivery capabilities, and they are widely used in drug delivery, gene therapy, cosmetics and other fields.

[0005] The literature "Effect of Preparation Temperature on Lipoic Acid Lipid Nanoparticles" describes how suitable lipid matrices and emulsifiers for lipoic acid (ALA) loading were obtained through lipid and emulsifier screening experiments. Based on this, lipoic acid lipid nanoparticles (ALA-NLCs) were prepared at 55℃ and 65℃ using high-pressure homogenization (HPH). The effects of preparation temperature and storage time on the properties of ALA-NLCs, such as ALA loading, particle size, stability, and morphology, were investigated using high-performance liquid chromatography (HPLC), dynamic light scattering (DLS), atomic force microscopy (AFM), and micro-differential scanning calorimetry (micro-DSC). The results showed that preparation temperature and storage time had a significant impact on particle size, zeta potential, and ALA loading.

[0006] Currently, research on lipoic acid liposomes for use in cosmetics is still very limited. It is of great significance to prepare lipoic acid liposomes through appropriate liposome preparation processes to improve the bioavailability of lipoic acid, significantly enhance its efficacy, and improve its compatibility with other functional ingredients. Summary of the Invention

[0007] In view of this, the purpose of this invention is to provide a lipoic acid liposome, its preparation method and application. By improving the raw material formulation and preparation process of the lipoic acid liposome, the product stability and safety of use are significantly improved, while the transdermal absorption performance is significantly improved, achieving long-lasting release. At the same time, it can load multiple components, significantly improving the whitening and anti-wrinkle effects of the product.

[0008] To achieve the above-mentioned objectives, the technical solution of this invention is as follows: On one hand, the present invention provides a lipoic acid liposome, wherein the raw materials of the lipoic acid liposome include: Thioctanoic acid, mixed solvents, mixed lipids, and stabilizers; The mixed lipids include hydrogenated lecithin, camellia seed oil, and tocopheryl acetate.

[0009] Preferably, the raw materials for the lipoic acid liposomes include, by weight, 1-5 parts of lipoic acid, 40-60 parts of mixed solvent, 20-50 parts of mixed lipids, and 1-5 parts of stabilizer.

[0010] More preferably, the raw materials for the lipoic acid liposomes include, by weight: 2-4 parts lipoic acid, 55-60 parts mixed solvent, 30-40 parts mixed lipids, and 3-5 parts stabilizer.

[0011] More preferably, the raw materials for the lipoic acid liposomes include, by weight: 3 parts lipoic acid, 58 parts mixed solvent, 35 parts mixed lipids, and 3 parts stabilizer.

[0012] Preferably, the raw material for the lipoic acid liposomes also includes Cystoseira malachite extract.

[0013] More preferably, the raw materials for the lipoic acid liposomes include, by weight: 1-5 parts lipoic acid, 0.1-2 parts of *Echinochloa crus-galli* extract, 40-60 parts mixed solvent, 20-50 parts mixed lipids, and 1-5 parts stabilizer.

[0014] More preferably, the raw materials for the lipoic acid liposomes include, by weight: 2-4 parts lipoic acid, 0.5-1 part *Echinops latifolius* extract, 55-60 parts mixed solvent, 30-40 parts mixed lipids, and 3-5 parts stabilizer.

[0015] Most preferably, the raw materials for the lipoic acid liposomes include, by weight: 3 parts lipoic acid, 1 part *Echinops latifolius* extract, 58 parts mixed solvent, 35 parts mixed lipids, and 3 parts stabilizer.

[0016] Preferably, the mixed solvent comprises 1,3-propanediol, glycerol, and water.

[0017] More preferably, the mass ratio of 1,3-propanediol, glycerol and water is 10-15:5-10:20-40, and more preferably 12:7:25.

[0018] More preferably, the mass ratio of hydrogenated lecithin, camellia seed oil and tocopheryl acetate is 5-20:20-40:1-2, and more preferably 10:25:1.5.

[0019] Preferably, the stabilizer is selected from at least one of polysorbate-80, phenoxyethanol, methylparaben, potassium sorbate, vitamin E, and lecithin, and more preferably a combination of polysorbate-80 and phenoxyethanol. More preferably, the mass ratio of polysorbate-80 to phenoxyethanol is 10-20:1, and most preferably 15:1.

[0020] Furthermore, the present invention provides a method for preparing the above-mentioned lipoic acid liposomes, comprising the following steps: (1) Dissolve the formula amount of glycerol, 1,3-propanediol and thioctic acid in an emulsifying pot by heating, add the formula amount of hydrogenated lecithin and disperse evenly, add water and disperse evenly to obtain phase A, cool and set aside; (2) The formula amounts of camellia seed oil, tocopheryl acetate, polysorbate-80, spirulina extract and phenoxyethanol were heated to obtain phase B; (3) Quickly inject phase B into the stirred phase A, heat and disperse to obtain a dispersion; (4) Pass the dispersion through a microjet once to obtain the lipoic acid liposomes.

[0021] Preferably, in step (1), the heating temperature is 50-80°C. More preferably, it is 65°C.

[0022] Preferably, in step (1), the temperature of the water is 50-80°C. More preferably, it is 65°C.

[0023] Preferably, in step (1), the cooling temperature is 20-40°C, and more preferably 35°C.

[0024] Preferably, in step (2), the heating temperature is 30-45°C, and more preferably 35°C.

[0025] Preferably, in step (3), the stirring speed is 400-1000 r / min, and more preferably 600 r / min.

[0026] Preferably, in step (3), the heating temperature is 30-45℃ and the time is 30-60min, and more preferably, the heating temperature is 35℃ and the time is 45min.

[0027] Preferably, in step (3), the dispersion is performed at a rotation speed of 10,000-20,000 rpm for 2-10 minutes; more preferably, it is performed at a rotation speed of 16,000 rpm for 5 minutes.

[0028] Preferably, in step (4), the pressure of the microjet is 15,000-25,000 psi, more preferably 18,000 psi.

[0029] As a specific example of the preparation method of the present invention, the preparation method includes the following steps: (1) The formula amount of glycerol, 1,3-propanediol and thioctic acid are heated to 65°C in an emulsifying pot and stirred until dissolved. The formula amount of hydrogenated lecithin is added and dispersed evenly. Deionized water at 65°C is added to the emulsifying pot and dispersed evenly to obtain phase A. The mixture is stirred and cooled to 35°C for later use. (2) The formula amounts of camellia seed oil, tocopheryl acetate, eugenol extract, and stabilizer are heated to 35°C in a water bath to obtain phase B; (3) Quickly inject phase B into phase A which is being stirred at high speed (600 r / min), stir in a water bath at 35°C for 45 min, and then disperse at a speed of 16,000 rps for 5 min to obtain the dispersion. (4) Pass the dispersion through a microjet once at a pressure of 18000 psi to obtain the lipoic acid liposomes.

[0030] Finally, the present invention provides the application of the above-mentioned lipoic acid liposomes in the preparation of cosmetics with whitening and anti-wrinkle effects.

[0031] Beneficial effects Compared with the prior art, the present invention has at least the following beneficial effects: This invention significantly improves product stability and safety by modifying the preparation process and raw material formulation of lipoic acid liposomes, while also significantly improving transdermal absorption performance and achieving effective release. Furthermore, it loads multiple components, significantly enhancing the product's whitening and anti-wrinkle effects. Attached Figure Description

[0032] Figure 1 This is an electron micrograph of the liposomes prepared in Example 4 of the present invention; Figure 2 The results are for the particle size detection of liposomes prepared in Example 4 of this invention. Detailed Implementation

[0033] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0034] In the following examples, the raw materials were all obtained through conventional commercial means.

[0035] Thioctic acid CAS number 1077-28-7; Tocopheryl acetate CAS number 7695-91-2.

[0036] The extract of *Eurysporium argenteum* can be prepared using commercially available products or conventional extraction processes. As an example, the extract of *Eurysporium argenteum* in the following embodiment is prepared by the following steps: *Eurysporium argenteum* is processed by a cell wall breaker, 10 times its weight of water is added, and the mixture is stirred and extracted at 65°C for 5 hours. The mixture is then filtered, concentrated, and freeze-dried to obtain the extract of *Eurysporium argenteum*.

[0037] Examples 1-10 Raw material formulation: alpha-lipoic acid, *Echinochloa crus-galli* extract, mixed solvents, mixed lipids, and stabilizers; Mixed lipids: Hydrogenated lecithin, camellia seed oil and tocopheryl acetate are mixed in a mass ratio of 10:25:1.5; Mixed solvent: 1,3-propanediol, glycerol and deionized water are mixed in a mass ratio of 12:7:25; Stabilizer: a combination of polysorbate-80 and phenoxyethanol in a mass ratio of 15:1.

[0038] The formulations and dosages for Examples 1-10 are as follows:

[0039] Examples 1-10 were all prepared according to the following steps: (1) The formula amount of glycerol, 1,3-propanediol and thioctic acid are heated to 65°C in an emulsifying pot and stirred until dissolved. The formula amount of hydrogenated lecithin is added and dispersed evenly. Deionized water at 65°C is added to the emulsifying pot and dispersed evenly to obtain phase A. The mixture is stirred and cooled to 35°C for later use. (2) The formula amounts of camellia seed oil, tocopheryl acetate, eugenol extract, and stabilizer are heated to 35°C in a water bath to obtain phase B; (3) Quickly inject phase B into phase A which is being stirred at high speed (600 r / min), stir in a water bath at 35°C for 45 min, and then disperse at a speed of 16,000 rps for 5 min to obtain the dispersion. (4) Pass the above dispersion through a microjet once at a pressure of 18000 psi to obtain the lipoic acid liposome.

[0040] Example 11 Unlike Example 4: In the mixed lipids, the mass ratio of hydrogenated lecithin, camellia seed oil and tocopheryl acetate was 5:40:1.

[0041] In the mixed solvent, the mass ratio of 1,3-propanediol, glycerol and water is 10:10:40.

[0042] Everything else is the same.

[0043] Example 12 Unlike Example 4: In the mixed lipids, the mass ratio of hydrogenated lecithin, camellia seed oil and tocopheryl acetate was 20:20:2.

[0044] In the mixed solvent, the mass ratio of 1,3-propanediol, glycerol and water is 15:5:20.

[0045] Everything else is the same. Comparative Example 1 Unlike Example 9: Mixed lipids: Soybean lecithin, camellia seed oil and tocopheryl acetate are mixed in a mass ratio of 10:25:1.5, that is, soy lecithin is used to replace hydrogenated lecithin in the preparation process.

[0046] Everything else is the same.

[0047] Comparative Example 2 Unlike Example 9: Mixed lipids: Hydrogenated lecithin and tocopheryl acetate are mixed in a mass ratio of 10:26.5, meaning that camellia seed oil is not added during the preparation process.

[0048] Everything else is the same.

[0049] Comparative Example 3 Unlike Example 9: Mixed lipids: Hydrogenated lecithin and camellia seed oil are mixed in a mass ratio of 10:26.5, meaning that no tocopheryl acetate is added during the preparation process.

[0050] Everything else is the same.

[0051] Comparative Example 4 Unlike Example 9: Mixed solvent: 1,3-butanediol, glycerol and water are mixed in a mass ratio of 12:7:25.

[0052] Everything else is the same.

[0053] Comparative Example 5 Unlike Example 4, this example omphalocele extract was not added; instead, an equal mass of nicotinamide was added. Everything else remained the same.

[0054] Comparative Example 6 Unlike Example 4, lipoic acid was not added to prepare *Strombyx mori* extract liposomes. Everything else was the same.

[0055] Result detection 1. Particle size detection Particle size was determined using a Baxter nanoparticle size potential analyzer. The average particle size results are as follows:

[0056] The results show that the average particle size of the liposomes prepared in the embodiments and comparative examples of the present invention is below 150 nm, which is more conducive to skin absorption and is suitable for use in cosmetics.

[0057] 2. Liposome stability test The liposome systems prepared in the examples and comparative examples were sealed and placed at room temperature (50±1℃). The appearance, layering, and precipitation were observed after 0 days and 30 days, and the pH was measured.

[0058] as follows:

[0059]

[0060] The results showed that the liposome system prepared in the examples exhibited good stability in the high-temperature accelerated experiment, while the comparative examples 1-4 had poor stability and were more likely to undergo lipid hydrolysis and release of lipoic acid during storage.

[0061] 3. Whitening effect test 3.1 Detection of Tyrosinase Inhibition Rate Preparation of sample solutions: Take the liposome systems prepared in the examples and comparative examples, dilute them 10 times with PBS solution (phosphate buffer), and mix well to obtain sample solutions.

[0062] Preparation of tyrosinase solution: Take tyrosinase (enzyme activity 2000U / mg) and prepare a 2U / mL tyrosinase solution with PBS.

[0063] Preparation of levodopa solution: Prepare a 2 mmol / L levodopa solution using PBS.

[0064] The reaction was carried out in a constant temperature water bath at 37°C for 10 minutes. The absorbance at 475 nm was measured using a microplate reader, and the OD value was read. The reaction system is as follows: ODx: 50 μL tyrosinase solution + 50 μL sample solution + 100 μL levodopa solution; OD0: 100 μL PBS solution + 100 μL levodopa solution; OD1: 50 μL tyrosinase solution + 50 μL PBS solution + 100 μL levodopa solution.

[0065] Inhibition rate (%) = [1 - (ODx - OD0) / (OD1 - OD0)] × 100% Each group was tested in triplicate, and the average value was taken. The results are as follows:

[0066] 3.2 Volunteer Experiment The liposome systems prepared in Examples 4, 9, 1, and 5 were formulated into serums with an addition amount of 1% according to the same conventional serum formula. The control group was not supplemented with liposome systems.

[0067] The volunteers were healthy women aged 30-40, randomly divided into 5 groups of 10 each. After cleansing their faces with water every morning and evening, they applied the sample essence to the areas with pigmentation on their faces. During this period, they did not use any other skin care products and strictly protected themselves from the sun.

[0068] Before using the serum, the melanin content was tested using an MDD4-Mexameter MX18 melanin analyzer and recorded as M0. After 30 days of use, the melanin content was tested again using the MDD4-Mexameter MX18 melanin analyzer and recorded as M1. After 60 days of use, the melanin content was tested again using the MDD4-Mexameter MX18 melanin analyzer and recorded as M2.

[0069] 30-day improvement rate: (M0-M1) / M0×100%; 60-day improvement rate: (M0-M2) / M0×100%.

[0070] The results are as follows:

[0071] The results showed that the zinc sulfate liposomes prepared in the embodiments of the present invention, or the liposomes prepared by combining zinc sulfate with *Strombus haematocephala* extract, could significantly improve tyrosinase inhibitory activity. Comparative Example 6 showed that the tyrosinase inhibitory activity was low after liposomes prepared from *Strombus haematocephala* extract at this dosage. The results of Examples 1-7 and 11-12 showed that the addition of *Strombus haematocephala* extract significantly improved the whitening effect of lipoic acid-only liposomes.

[0072] Meanwhile, volunteer experiments showed that the liposomes prepared in the examples, when applied to serums, could significantly reduce melanin content and improve whitening effects. Furthermore, comparative results indicated that the choice of mixed lipids has a crucial impact on the release and absorption of active ingredients in the final liposomes. For the complex active ingredients of lipoic acid and *Alternanthera philoxeroides* extract, hydrogenated lecithin, camellia seed oil, and tocopheryl acetate were more conducive to the release and absorption of active ingredients in the skin; in comparison, the effect of the solvent was relatively small.

[0073] 4. Anti-wrinkle effect test The experimental method was the same as described in section 3.2, Volunteer Experiment. The application area was selected as a 5×5cm area on both sides of the face. The skin elasticity parameter R2 value (which reflects skin elasticity; the higher the R2 value, the better the skin elasticity) was tested on the left side of the face before using the serum, after 30 days of use, and after 60 days of use.

[0074] Before using the serum, the skin elasticity parameter R2 value was tested using a Reviscometer RVM600 skin elasticity tester and recorded as A0. After 30 days of use, the skin elasticity parameter R2 value was tested again using the Reviscometer RVM600 skin elasticity tester and recorded as A1. After 60 days of use, the skin elasticity parameter R2 value was tested again using the Reviscometer RVM600 skin elasticity tester and recorded as A2.

[0075] 30-day improvement rate: (A1-A0) / A0×100%; 60-day improvement rate: (A2-A0) / A0×100%.

[0076] The results are as follows:

[0077] The results showed that the liposomes prepared in Example 4 of this invention, when applied to a serum, could significantly improve skin elasticity and promote wrinkle repair. Meanwhile, Comparative Example 1 indicated that the selection of the mixed lipids has a crucial impact on the release and absorption of the active ingredients in the final liposomes. For the complex active ingredients of lipoic acid and *Alternanthera philoxeroides* extract, hydrogenated lecithin, camellia seed oil, and tocopheryl acetate were more conducive to the release and absorption of the active ingredients in the skin. Comparative Example 5 showed that the addition of *Alternanthera philoxeroides* extract significantly improved the anti-wrinkle effect of the lipoic acid-only liposomes.

[0078] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A lipoic acid liposome, characterized in that, The raw materials for the lipoic acid liposomes include, by weight: 1-5 parts lipoic acid, 40-60 parts mixed solvent, 20-50 parts mixed lipids, and 1-5 parts stabilizer. The mixed lipids include hydrogenated lecithin, camellia seed oil, and tocopheryl acetate; The mixed solvent comprises 1,3-propanediol, glycerol, and water in a mass ratio of 10-15:5-10:20-40.

2. The lipoic acid liposome according to claim 1, characterized in that, The raw materials for the lipoic acid liposomes, by weight, include: 3 parts lipoic acid, 58 parts mixed solvent, 35 parts mixed lipid and 3 parts stabilizer.

3. The lipoic acid liposome according to claim 1, characterized in that, It also includes extracts from *Eurysporium argenteum*.

4. The lipoic acid liposome according to claim 3, characterized in that, The raw materials for the lipoic acid liposomes, by weight, include: 1-5 parts lipoic acid, 0.1-2 parts of *Echinochloa crus-galli* extract, 40-60 parts mixed solvent, 20-50 parts mixed lipids, and 1-5 parts stabilizer.

5. The lipoic acid liposome according to claim 4, characterized in that, The raw materials for the lipoic acid liposomes, by weight, include: 2-4 parts lipoic acid, 0.5-1 part of *Echinochloa crus-galli* extract, 55-60 parts mixed solvent, 30-40 parts mixed lipids, and 3-5 parts stabilizer.

6. The lipoic acid liposome according to claim 5, characterized in that, The raw materials for the lipoic acid liposomes, by weight, include: 3 parts lipoic acid, 1 part *Eucalyptus globulus* extract, 58 parts mixed solvent, 35 parts mixed lipids, and 3 parts stabilizer.

7. The lipoic acid liposome according to claim 6, characterized in that, The mass ratio of 1,3-propanediol, glycerol, and water is 12:7:

25.

8. The lipoic acid liposome according to any one of claims 1-7, characterized in that, The mass ratio of hydrogenated lecithin, camellia seed oil and tocopheryl acetate is 5-20:20-40:1-2.

9. The lipoic acid liposome according to claim 8, characterized in that, The mass ratio of hydrogenated lecithin, camellia seed oil, and tocopheryl acetate is 10:25:1.

5.

10. The lipoic acid liposome according to any one of claims 1-7, characterized in that, The stabilizer is a combination of polysorbate-80 and phenoxyethanol.

11. The lipoic acid liposome according to claim 10, characterized in that, The mass ratio of polysorbate-80 to phenoxyethanol is 10-20:

1.

12. The method for preparing lipoic acid liposomes according to any one of claims 4-11, characterized in that, Includes the following steps: (1) Dissolve the formula amount of glycerol, 1,3-propanediol and thioctic acid in an emulsifying pot by heating, add the formula amount of hydrogenated lecithin and disperse evenly, add water and disperse evenly to obtain phase A, cool and set aside; (2) Heat the prescribed amounts of camellia seed oil, tocopheryl acetate, eugenol extract, and stabilizer to obtain phase B; (3) Quickly inject phase B into the stirred phase A, heat and disperse to obtain a dispersion; (4) Pass the dispersion through a microjet once to obtain the lipoic acid liposomes.

13. The method for preparing lipoic acid liposomes according to claim 12, characterized in that... ； In step (1), the temperature of the water is 50-80℃, and the cooling temperature is 20-40℃; In step (2), the heating temperature is 30-45℃; In step (3), the stirring speed is 400-1000 r / min, the heating temperature is 30-45℃, the time is 30-60 min, and the dispersion is: dispersed at a speed of 10,000-20,000 rps for 2-10 min; In step (4), the pressure of the microjet is 15,000-25,000 psi.

14. The use of the lipoic acid liposomes according to any one of claims 1-11 in the preparation of cosmetics with whitening and anti-wrinkle effects.

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