Lipoic acid liposome as well as preparation method and application thereof

Abstract

The invention relates to the technical field of liposome preparation, in particular to lipoic acid liposome as well as a preparation method and application thereof. The lipoic acid liposome is prepared from the following raw materials: lipoic acid, a mixed solvent, mixed lipid and a stabilizer, wherein the mixed lipid comprises hydrogenated lecithin, camellia seed oil and tocopheryl acetate. By improving the raw material formula and the preparation process of the lipoic acid liposome, the stability and the use safety of the product are remarkably improved, meanwhile, the transdermal absorption performance is remarkably improved, long-acting release is realized, multiple components are loaded at the same time, and the whitening and anti-wrinkle effects of the product are remarkably improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of liposome preparation, and particularly relates to a lipoic acid liposome, a preparation method and application thereof. BACKGROUND

[0002] Alpha-lipoic acid (ALA) is a powerful all-purpose antioxidant, often added in vitamins and anti-aging agents. But it still has great application limitations in practical application.

[0003] The lipoic acid has the following defects: (1) the lipoic acid is extremely unstable, and is very sensitive to light, heat and oxygen, and is prone to degradation and dimerization, resulting in rapid loss of antioxidant activity. Related products will quickly turn yellow and lose efficacy under light, and the active ingredients will be largely degraded during the production process (such as high-temperature sterilization, emulsification) or high-temperature storage in summer, and will gradually deteriorate after being opened and contacted with oxygen in the air, resulting in a short shelf life of related products; (2) high-concentration L-lipoic acid can cause discomfort such as tingling, heating and redness on human skin, and its strong acidity and possible effect on certain skin receptors or nerve endings require higher tolerance; (3) the transdermal absorption rate of lipoic acid is low, and a large amount of active ingredients remain on the surface of the skin when applied to the skin, and cannot penetrate into the target target point (such as the dermis) to play the corresponding effect; (4) poor formulation compatibility, the lipoic acid is active and prone to interact with other ingredients (such as certain metal ions, other active agents) in the formula, resulting in instability (such as discoloration, delamination, and viscosity change) of the entire formula system, limiting its compounding use. Chinese patent CN101721369A discloses a preparation method of alpha-lipoic acid liposome. The method is to dissolve soybean phospholipid and cholesterol in an organic solvent, then add to an alpha-lipoic acid solution to form a liposome solution, the mass ratio of soybean phospholipid, cholesterol and alpha-lipoic acid is 3.1~3.5:1:0.03~0.06, a film is formed by water bath, reduced pressure and rotary evaporation, an auxiliary agent diethyl ether is added, and uniform liposomes are formed after hydration and crushing. The lipoic acid liposome prepared by the preparation method has uniformity, stability and high encapsulation efficiency, and is beneficial to industrial production.

[0004] Liposomes are small spherical carriers composed of phospholipid bilayers, and their core advantages lie in biomimetic structure and multifunctional drug loading capacity, and they are widely used in drug delivery, gene therapy, cosmetics and other fields.

[0005] In the document "Effect of preparation temperature on liposomes of lipoic acid", a lipid matrix and an emulsifier suitable for lipoic acid (ALA) loading are obtained through lipid and emulsifier screening experiments, and lipoic acid liposomes (ALA-NLC) are prepared at 55 DEG C and 65 DEG C respectively by high pressure homogenization (HPH). The effects of preparation temperature and storage time on the properties of ALA-NLC such as ALA loading, particle size, stability and morphology are studied by high performance liquid chromatography (HPLC), dynamic light scattering (DLS), atomic force microscopy (AFM), micro-differential scanning calorimetry (micro-DSC). The results show that the preparation temperature and storage time have great influence on the particle size, zeta potential and ALA loading.

[0006] At present, the research on lipoic acid liposomes for cosmetic use is still very scarce. It is of great significance to prepare lipoic acid liposomes through a suitable liposome preparation process to improve the bioavailability of lipoic acid, significantly improve its efficacy, and improve the compatibility with other efficacy ingredients. SUMMARY

[0007] Therefore, the purpose of the present application is to provide a lipoic acid liposome and a preparation method and application thereof. By improving the raw material formula and preparation process of the lipoic acid liposome, the product stability and use safety are significantly improved, the transdermal absorption performance is significantly improved, long-acting release is realized, multiple components are loaded, and the whitening and anti-wrinkle efficacy of the product is significantly improved.

[0008] To achieve the above-mentioned purposes, the technical solutions of the present application are as follows: In one aspect, the present application provides a lipoic acid liposome, the raw materials of the lipoic acid liposome comprising: lipoic acid, mixed solvent, mixed lipid and stabilizer. The mixed lipid comprises hydrogenated lecithin, camellia seed oil and tocopheryl acetate.

[0009] Preferably, the raw materials of the lipoic acid liposome comprise, by weight: 1-5 parts of lipoic acid, 40-60 parts of mixed solvent, 20-50 parts of mixed lipid and 1-5 parts of stabilizer.

[0010] Further preferably, the raw materials of the lipoic acid liposome comprise, by weight: 2-4 parts of lipoic acid, 55-60 parts of mixed solvent, 30-40 parts of mixed lipid and 3-5 parts of stabilizer.

[0011] Further preferably, the raw materials of the lipoic acid liposome comprise, by weight: 3 parts of lipoic acid, 58 parts of mixed solvent, 35 parts of mixed lipid and 3 parts of stabilizer.

[0012] Preferably, the raw material of the liposome of lipoic acid further comprises Cystoseira tamariscafolia extract.

[0013] Further preferably, the raw material of the liposome of lipoic acid comprises, by weight: lipoic acid 1-5 parts, Cystoseira tamariscafolia extract 0.1-2 parts, mixed solvent 40-60 parts, mixed lipids 20-50 parts, and stabilizer 1-5 parts.

[0014] Further preferably, the raw material of the liposome of lipoic acid comprises, by weight: lipoic acid 2-4 parts, Cystoseira tamariscafolia extract 0.5-1 part, mixed solvent 55-60 parts, mixed lipids 30-40 parts, and stabilizer 3-5 parts.

[0015] Most preferably, the raw material of the liposome of lipoic acid comprises, by weight: lipoic acid 3 parts, Cystoseira tamariscafolia extract 1 part, mixed solvent 58 parts, mixed lipids 35 parts, and stabilizer 3 parts.

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

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

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

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

[0020] In another aspect, the present application provides a preparation method of the above-mentioned liposome of lipoic acid, comprising the following steps: (1) Dissolve the formula amount of glycerol, 1,3-propanediol, and lipoic acid in an emulsifying pot, add the formula amount of hydrogenated lecithin and disperse uniformly, add water and disperse uniformly to obtain phase A, and cool for standby; (2) Heat the formula amount of camellia seed oil, tocopheryl acetate, polysorbate-80, Cystoseira tamariscafolia extract, and phenoxyethanol to obtain phase B; (3) Rapidly inject phase B into the stirring phase A, heat, disperse, and obtain a dispersion material; (4) Pass the dispersion material through a microjet once to obtain the liposome of lipoic acid.

[0021] Preferably, in step (1), the temperature of heating is 50-80℃. Further preferably, it is 65℃.

[0022] Preferably, in step (1), the temperature of heating is 50-80℃. Further preferably, it is 65℃.

[0023] Preferably, in step (1), the temperature of cooling is 20-40℃. Further preferably, it is 35℃.

[0024] Preferably, in step (2), the temperature of heating is 30-45℃. Further preferably, it is 35℃.

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

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

[0027] Preferably, in step (3), the dispersing is carried out at a speed of 10-20thousands rps for 2-10min. Further preferably, it is carried out at a speed of 16thousands rps for 5min.

[0028] Preferably, in step (4), the pressure of microjet is 15000-25000psi. Further preferably, it is 18000psi.

[0029] As a specific example of the preparation method of the present application, the preparation method comprises the following steps: (1) Formula amount of glycerin, 1,3-propanediol, lipoic acid is heated to 65℃ in an emulsifying kettle and stirred until dissolved. Formula amount of hydrogenated lecithin is added and dispersed uniformly. Deionized water at 65℃ is added into the emulsifying kettle and dispersed uniformly to obtain phase A. Stirring is carried out to cool to 35℃ for standby; (2) Formula amount of camellia seed oil, tocopheryl acetate, N. oceanica extract, and stabilizer are heated to 35℃ in a water bath to obtain phase B; (3) Phase B is quickly injected into phase A which is stirred at a high speed (600r / min) to obtain a dispersed material, and then stirred in a 35℃ water bath for 45min. The dispersed material is dispersed at a speed of 16thousands rps for 5min to obtain a dispersed material body; (4) The dispersed material body is passed through a microjet once at a pressure of 18000psi to obtain the lipoic acid liposome.

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

[0031] Beneficial effects Compared with the prior art, the application has at least the following beneficial effects: The application significantly improves the product stability, use safety, transdermal absorption performance, and the product whitening and anti-wrinkle effects by improving the preparation process and raw material formula of the liposome of lipoic acid. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 The electron microscope image of the liposome prepared in Example 4 of the application; Figure 2 The particle size detection result of the liposome prepared in Example 4 of the application. DETAILED DESCRIPTION

[0033] The following non-limiting examples can make those skilled in the art more fully understand the application, but do not limit the application in any way. The following content is only an exemplary description of the scope of the application claimed by the application, and those skilled in the art can make various changes and modifications to the application according to the disclosed content, and it should also belong to the scope of the application claimed by the application.

[0034] In the following examples, the raw materials are obtained by conventional commercial channels.

[0035] Lipoic acid CAS No. 1077-28-7; Tocopherol acetate CAS No. 7695-91-2.

[0036] The Euglena gracilis extract can be prepared using commercially available products or conventional extraction processes. As an example, the Euglena gracilis extract in the following examples is prepared by the following steps: treating Euglena gracilis with a cell wall breaking machine, adding 10 times the weight of water, stirring at 65°C for 5h, filtering, concentrating, and freeze-drying to obtain the Euglena gracilis extract.

[0037] Examples 1-10 Raw material formula: lipoic acid, Euglena gracilis extract, mixed solvent, mixed lipid, and stabilizer; Mixed lipid: hydrogenated lecithin, camellia seed oil, and tocopherol 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: polysorbate-80 and phenoxyethanol are combined in a mass ratio of 15:1.

[0038] The dosage of the formula of Examples 1-10 is as follows:

[0039] Examples 1-10 are prepared according to the following steps: (1) The formula amount of glycerol, 1,3-propanediol, lipoic acid is heated to 65℃ in an emulsifying kettle and stirred until dissolved, and the formula amount of hydrogenated lecithin is added and uniformly dispersed; deionized water at 65℃ is added to the emulsifying kettle and uniformly dispersed to obtain phase A, which is stirred and cooled to 35℃ for standby; (2) The formula amount of camellia seed oil, tocopherol acetate, Nannochloropsis oceanica extract, and stabilizer is heated to 35℃ in a water bath to obtain phase B; (3) Phase B is quickly injected into phase A under high-speed stirring (600 r / min), and stirred in a 35℃ water bath for 45 min, and then dispersed at a speed of 16000 rps for 5 min to obtain a dispersion material; (4) The dispersion material is passed through a micro-jet once at a pressure of 18000 psi to obtain the lipoic acid liposome.

[0040] Example 11 The difference from Example 4 is that: The mass ratio of hydrogenated lecithin, camellia seed oil, and tocopherol acetate in the mixed lipid is 5:40:1.

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

[0042] The rest are the same.

[0043] Example 12 The difference from Example 4 is that: The mass ratio of hydrogenated lecithin, camellia seed oil, and tocopherol acetate in the mixed lipid is 20:20:2.

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

[0045] The rest are the same. Comparative Example 1 The difference from Example 9 is that: The mixed lipid: soybean phospholipid, camellia seed oil, and tocopherol acetate are mixed in a mass ratio of 10:25:1.5, i.e., soybean phospholipid is used to replace hydrogenated lecithin in the preparation process.

[0046] The rest are the same.

[0047] Comparative Example 2 The difference from Example 9 is that: The mixed lipid: hydrogenated lecithin and tocopherol acetate are mixed in a mass ratio of 10:26.5, i.e., camellia seed oil is not added in the preparation process.

[0048] The rest are the same.

[0049] Comparative Example 3 Different from example 9 is that: Mixed lipid: hydrogenated lecithin, camellia seed oil mixed in a mass ratio of 10:26.5, that is, no tocopherol acetate is added in the preparation process.

[0050] The rest are the same.

[0051] Comparative example 4 Different from example 9 is that: Mixed solvent: 1,3-butanediol, glycerol and water mixed in a mass ratio of 12:7:25.

[0052] The rest are the same.

[0053] Comparative example 5 Different from example 4 is that no eustigmatophyta extract is added, and an equivalent amount of nicotinamide is added instead. The rest are the same.

[0054] Comparative example 6 Different from example 4 is that no thioctic acid is added, and eustigmatophyta extract liposomes are prepared. The rest are the same.

[0055] Result detection 1, Particle size detection The particle size is determined by using a Betatau nanoparticle size potential analyzer, and the average particle size detection results are as follows:

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

[0057] 2, Liposome stability detection The liposome system prepared in the examples and comparative examples is sealed and placed in a room temperature 50±1℃ environment, and the appearance, layering and precipitation are observed after 0 days and 30 days, and the pH is measured.

[0058] As follows:

[0059]

[0060] The results show that the liposome system prepared in the examples exhibits good stability in the high temperature acceleration experiment, and the comparative examples 1-4 have poor stability, and the hydrolysis of lipids and the release of thioctic acid are more likely to occur during storage.

[0061] 3, Whitening effect detection 3.1 Tyrosinase inhibition rate detection Preparation of sample solution: the liposome system prepared in the examples and the comparative examples was diluted 10 times with PBS solution (phosphate buffer solution), and the sample solution was obtained after mixing.

[0062] Preparation of tyrosinase solution: tyrosinase (enzyme activity 2000 U / mg) was prepared into a 2 U / mL tyrosinase solution with PBS.

[0063] Preparation of levodopa solution: levodopa was prepared into a 2 mmol / L levodopa solution with PBS.

[0064] The reaction was carried out in a constant temperature water bath, the temperature was maintained at 37°C, the reaction was carried out for 10 min, the absorbance at 475 nm was measured with an enzyme marker, and the OD value was read. The reaction system was 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)] x 100% Each group was tested in triplicate, and the average value was taken, and the results were as follows:

[0066] 3.2 Volunteer experiment The liposome system prepared in Example 4, Example 9, Comparative Example 1 and Comparative Example 5 was respectively prepared into a 1% added amount of serum according to the same conventional serum formula, and was prepared according to the conventional method, and the control group did not add the liposome system.

[0067] The volunteers were healthy women aged 30-40 years old, and were randomly divided into 5 groups, 10 people in each group. After washing the face with water in the morning and evening, the sample serum was used on the pigmented area of the face, no other skin care products were used during the period, and strict sun protection was carried out.

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

[0069] 30-day improvement rate: (M0 - M1) / M0 x 100%; 60 days improvement rate: (A2-A0) / A0x100%.

[0070] Results are as follows:

[0071] Results show that the zinc sulfate liposome or zinc sulfate combined with eustigmatophyte extract liposome prepared by the embodiment of the application can significantly improve the tyrosinase inhibitory activity. Comparative Example 6 shows that the tyrosinase inhibitory activity is low after the eustigmatophyte extract is prepared into liposomes at this dosage. Results of Examples 1-7 and 11-12 show that the addition of eustigmatophyte extract significantly improves the whitening effect of liposomes of a single component of thioctic acid.

[0072] At the same time, the volunteer experiment shows that the liposomes prepared in the embodiment can significantly reduce the melanin content and improve the whitening effect when applied to the essence. At the same time, the results of the comparative example show that the selection of mixed lipids has a key influence on the release and absorption of active ingredients in the final liposomes. For the complex active ingredients of thioctic acid and eustigmatophyte extract, hydrogenated lecithin, camellia seed oil and tocopheryl acetate as lipids are more conducive to the release and absorption of active ingredients in the skin. In comparison, the influence of the solvent is smaller.

[0073] 4. Anti-wrinkle effect detection The experimental method is the same as that described in the volunteer experiment part of 3.2. The application area is selected as a 5x5 cm area on both sides of the face. The skin elasticity parameter R2 value (reflecting skin elasticity, the higher the R2 value, the better the skin elasticity) is tested before using the essence, 30 days after using the essence and 60 days after using the essence on the left side of the face.

[0074] Before using the essence, the skin elasticity parameter R2 value is tested by the skin elasticity tester Reviscometer RVM600, and recorded as A0. After using the essence for 30 days, the skin elasticity parameter R2 value is tested again by the skin elasticity tester Reviscometer RVM600, and recorded as A1. After using the essence for 60 days, the skin elasticity parameter R2 value is tested again by the skin elasticity tester Reviscometer RVM600, and recorded as A2.

[0075] 30 days improvement rate: (A1-A0) / A0x100%; 60 days improvement rate: (A2-A0) / A0x100%.

[0076] Results are as follows:

[0077] The results show that the liposome prepared in Example 4 is applied to the essence, which can significantly improve the skin elasticity and promote the wrinkle repair. Meanwhile, the results of Comparative Example 1 show that the selection of mixed lipids has a key influence on the release and absorption of active ingredients in the final liposome, and for the complex active ingredients of lipoic acid and euglena extract, hydrogenated lecithin, camellia seed oil and tocopheryl acetate as lipids are more conducive to the release and absorption of active ingredients in the skin. The results of Comparative Example 5 show that the addition of euglena extract significantly improves the anti-wrinkle effect of lipoic acid single-component liposome.

[0078] The above merely describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A lipoic acid liposome, characterized in that, The raw materials for the lipoic acid liposomes include: lipoic acid, mixed solvent, mixed lipids, and stabilizers; The mixed lipids include hydrogenated lecithin, camellia seed oil, and tocopheryl acetate.

2. The lipoic acid liposome according to claim 1, characterized in that, 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.

3. The lipoic acid liposome according to claim 2, 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.

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

5. The lipoic acid liposome according to claim 4, 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.

6. The lipoic acid liposome according to claim 5, 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.

7. The lipoic acid liposome according to claim 6, 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.

8. The lipoic acid liposome according to any one of claims 1-7, characterized in that, The mixed solvent comprises 1,3-propanediol, glycerol, and water.

9. The lipoic acid liposome according to claim 8, characterized in that, The mass ratio of 1,3-propanediol, glycerol and water is 10-15:5-10:20-40.

10. The lipoic acid liposome according to claim 9, characterized in that, The mass ratio of 1,3-propanediol, glycerol, and water is 12:7:

25.

11. 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.

12. The lipoic acid liposome according to claim 11, characterized in that, The mass ratio of hydrogenated lecithin, camellia seed oil, and tocopheryl acetate is 10:25:1.

5.

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

14. The lipoic acid liposome according to claim 13, characterized in that, The mass ratio of polysorbate-80 to phenoxyethanol is 10-20:

1.

15. The method for preparing lipoic acid liposomes according to any one of claims 8-14, 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.

16. The method for preparing lipoic acid liposomes according to claim 15, characterized in that, Preferably, in step (1), the heating temperature is 50-80℃; 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.

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

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