Nano astaxanthin liposome as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of cosmetics, and discloses a nano astaxanthin liposome as well as a preparation method and application thereof. The preparation method of the nano astaxanthin liposome comprises the following steps: dissolving a haematococcus pluvialis extract, lecithin and cholesterol in an organic solvent, heating to 35-40 DEG C for reaction, and performing vacuum condensation to remove the organic solvent to obtain a uniform astaxanthin-containing lipid film; and then adding a phosphoric acid buffer salt solution and tween-80, hydrating and dissolving, and then carrying out ultrasonic treatment to obtain the nano astaxanthin liposome. The nano astaxanthin liposome is uniform in particle size, good in dispersity, high in encapsulation efficiency and stability, outstanding in antioxidant activity, simple in preparation method, safe and non-toxic, and can be applied to preparation of cosmetics and large-scale production.

Description

Technical Field

[0001] The present invention relates to the field of cosmetics, and more specifically, to a nano astaxanthin liposome and a preparation method and application of the nano astaxanthin liposome. Background Art

[0002]

[0003] Astaxanthin (ASX) is a red, fat-soluble carotenoid that belongs to the same class of xanthophyll as other carotenoids such as β-cryptoxanthin, β-carotene, lycopene, and zeaxanthin. It is produced by various microorganisms and marine animals. The central non-polar region of astaxanthin consists of 13 bonds, which gives astaxanthin the ability to remove high-energy electrons. The hydroxyl and ketone groups on the two rings increase the polarity of astaxanthin, greatly enhancing its ability to pass through cell membranes. These unique chemical properties give astaxanthin some advantages related to biological activity, such as higher antioxidant activity than other carotenoids. However, the "polyene chain" in the middle of the astaxanthin structure makes it highly fat-soluble and insoluble in water; it is also easily decomposed by light and has poor stability, which greatly limits its application in cosmetics. Therefore, it is imperative to develop a carrier system that can improve its solubility and stability.

[0004] Liposomes are bilayer closed microvesicles with a biological membrane structure formed by phospholipids. They are hydrophilic and hydrophobic, and can encapsulate both water-soluble substances and fat-soluble substances. The membrane material of liposomes is generally natural raw materials, which have high biocompatibility and safety advantages. Liposome encapsulation can increase the solubility of astaxanthin in aqueous solution. Astaxanthin can penetrate the membrane of liposomes and interact with the polar groups of the membrane through hydrogen bonds. Therefore, the preparation of astaxanthin liposomes using liposome carrier technology can solve the water solubility problem of astaxanthin and improve the effective utilization rate of astaxanthin.

[0005] For example, Chinese patent CN110558435A discloses an astaxanthin nanoliposome and its preparation method and application. The preparation method dissolves astaxanthin, cholesterol, and phospholipids in an organic solvent, removes the organic solvent by reduced pressure evaporation to form a thin film, and then adds a buffer solution to dissolve and disperse. The invention has excellent characterization characteristics, a high synthesis rate, and a simple process. However, the film formed in the synthesis process of the invention has a certain wall hanging phenomenon, and the loss rate is large, so the utilization rate of astaxanthin is reduced.

[0006] For example, Chinese patent CN117017802A discloses a preparation method: astaxanthin and a cationic precursor are mixed in a solvent to react to obtain supramolecular astaxanthin; the supramolecular astaxanthin, soybean lecithin and a surfactant are reacted to obtain the supramolecular astaxanthin liposome. The preparation method has simple synthesis steps, convenient post-processing, high purity of the obtained product, and improved solubility and transdermal penetration of astaxanthin. However, the stability of the astaxanthin liposomes prepared by this method needs to be studied, and there is also the problem of astaxanthin loss during the preparation process.

[0007] Therefore, it is imperative to develop a nano-astaxanthin liposome that can be used in the cosmetics field and has good stability, high solubility, good antioxidant activity, safety and non-toxicity. Summary of the invention

[0008] Based on the problems existing in the prior art, the present invention aims to provide an astaxanthin liposome and a preparation method and application thereof. The key raw material used is astaxanthin, which is derived from natural Haematococcus pluvialis extract, which is recognized as the most abundant natural raw material for astaxanthin in the world. The nano astaxanthin liposome prepared by the method of the present invention can improve the solubility, stability and antioxidant activity of astaxanthin, and is easy to operate, easy to detect the encapsulation effect, safe and non-toxic, and improves the bioavailability of astaxanthin.

[0009] To achieve the above object, the technical solution adopted by the present invention is:

[0010] In one aspect, the present invention provides a method for preparing astaxanthin liposomes, comprising the following steps:

[0011] (1) dissolving a Haematococcus pluvialis extract, lecithin and cholesterol in an organic solvent and reacting them under heating conditions to obtain a lipid film containing astaxanthin;

[0012] (2) adding a hydrating solvent and Tween-80 to the lipid film obtained in step (1), and subjecting the mixture to ultrasound to obtain the nano-astaxanthin liposomes.

[0013] The mass ratio of the Haematococcus pluvialis extract, lecithin and cholesterol in step (1) is 20-50:50-250:10-50;

[0014] Preferably, the mass ratio of the Haematococcus pluvialis extract, lecithin and cholesterol in step (1) is 30-50:100-250:20-50;

[0015] More preferably, the mass ratio of the Haematococcus pluvialis extract, lecithin and cholesterol in step (1) is 30-40:150-250:30-50;

[0016] Further preferably, the mass ratio of the Haematococcus pluvialis extract, lecithin and cholesterol in step (1) is 30-40:200-250:40-50;

[0017] More preferably, the mass ratio of the Haematococcus pluvialis extract, lecithin and cholesterol in step (1) is 40:250:50.

[0018] The organic solvent in step (1) is anhydrous ethanol and / or chloroform;

[0019] Preferably, the organic solvent in step (1) is anhydrous ethanol.

[0020] The organic solvent is added until the Haematococcus pluvialis extract, lecithin and cholesterol are completely dissolved;

[0021] The heating temperature in step (1) is 35°C-40°C.

[0022] The hydration solvent in step (2) is a phosphate buffer solution with a pH of 6.5-7.5 or pure water;

[0023] Preferably, the hydration solvent in step (2) is phosphate buffer.

[0024] The volume ratio of the total mass of the Haematococcus pluvialis extract, lecithin and cholesterol described in step (1) to the hydration solvent and Tween-80 described in step (2) is 80-340 mg:10-50 mL:1-15 mL;

[0025] Preferably, the volume ratio of the total mass of the Haematococcus pluvialis extract, lecithin and cholesterol in step (1) to the hydration solvent and Tween-80 in step (2) is 160-340 mg: 10-50 mL: 1-15 mL;

[0026] Preferably, the volume ratio of the total mass of the Haematococcus pluvialis extract, lecithin and cholesterol in step (1) to the hydration solvent and Tween-80 in step (2) is 220-340 mg: 10-50 mL: 1-15 mL;

[0027] Further preferably, the volume ratio of the total mass of the Haematococcus pluvialis extract, lecithin and cholesterol in step (1) to the hydration solvent and Tween-80 in step (2) is 340 mg:50 mL:10 mL.

[0028] Preferably, the specific steps of the reaction described in step (1) are: dissolving the Haematococcus pluvialis extract, lecithin and cholesterol in an organic solvent and mixing them evenly, heating to 35° C.-40° C. for rotary evaporation, and vacuum condensing to remove the organic solvent to obtain a uniform astaxanthin-containing lipid film.

[0029] During the implementation of the present invention, it was found that after the astaxanthin-containing lipid film was hydrated and ultrasonically treated with only a hydration solvent, a certain wall hanging phenomenon still existed. Therefore, Tween-80 was added to the hydration solvent in the present invention. By controlling the volume ratio of the hydration solvent and Tween-80, the solubility of the astaxanthin lipid film can be significantly improved, thereby eliminating the wall hanging phenomenon.

[0030] As a preferred embodiment, 250 mg of lecithin, 50 mg of cholesterol and 40 mg of Haematococcus pluvialis extract are fully dissolved in 100 mL of anhydrous ethanol, transferred to a rotary evaporator, heated to 35°C-40°C for rotary evaporation, and vacuum condensed to remove the organic solvent to obtain a uniform lipid film containing astaxanthin. Subsequently, 50 mL of phosphate buffered saline and 10 mL of Tween-80 are added for hydration and dissolution, and then ultrasonic cell crusher is used for ultrasonication to obtain the astaxanthin liposomes.

[0031] The invention also provides nano astaxanthin liposomes prepared by the method.

[0032] The present invention also provides application of the nano-astaxanthin liposome obtained by the preparation method in the field of cosmetics.

[0033] Studies have shown that under different conditions, the stability of the nano-astaxanthin lipids prepared in this application has been greatly improved, and it is safe and non-toxic to the human body, and can be better used in cosmetics.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) The prepared nano-astaxanthin liposomes avoid the problem of astaxanthin loss during the preparation process, increase the solubility of astaxanthin, and improve the bioavailability of astaxanthin.

[0036] (2) The prepared nano-astaxanthin liposome system is stable, has good antioxidant activity, high encapsulation efficiency, uniform particle size, and good dispersibility.

[0037] (3) The prepared nano-astaxanthin liposomes have good stability for use in the cosmetics field.

[0038] (4) The prepared nano-astaxanthin liposomes are safe and non-toxic.

[0039] (5) The prepared nano-astaxanthin liposomes have a simple preparation process and are suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 UV absorption images of astaxanthin solutions with different concentrations;

[0041] Figure 2 This is the standard curve image for detecting the encapsulation efficiency of nano-astaxanthin liposomes;

[0042] Figure 3 The particle size data image of nano-astaxanthin liposomes prepared in Example 1-8;

[0043] Figure: A is Example 1; B is Example 2; C is Example 3; E is Example 4; F is Example 6; G is Example 7; H is Example 8;

[0044] Figure 4 The particle size data image of nano-astaxanthin liposomes prepared in Comparative Examples 1-4;

[0045] Figure: a is comparative example 1; b is comparative example 2; c is comparative example 3; d is comparative example 4;

[0046] Figure 5 This is the DPPH free radical scavenging rate image of the nano-astaxanthin liposomes prepared in Example 1-8;

[0047] Figure: A is Example 1; B is Example 2; C is Example 3; E is Example 4; F is Example 6; G is Example 7; H is Example 8;

[0048] Figure 6 The DPPH free radical scavenging rate image of the nano-astaxanthin lipid prepared in Comparative Examples 1-4;

[0049] Figure: a is comparative example 1; b is comparative example 2; c is comparative example 3; d is comparative example 4;

[0050] Figure 7 The stability photos of nano-astaxanthin liposomes under different storage conditions;

[0051] Figure 8 The stability photos of astaxanthin essence water under different storage conditions;

[0052] Fig. 9 Photos showing the stability of astaxanthin cream under different storage conditions. DETAILED DESCRIPTION

[0053] The technical scheme of the present invention is clearly and completely described below with specific implementation methods. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The materials, reagents, etc. used, if not otherwise specified, are reagents and materials available from commercial sources.

[0054] The manufacturers and product numbers of the reagents used in the following examples are as follows:

[0055] Reagents factory Part Number Haematococcus pluvialis extract Shanxi Zizhe Biological 472-61-7 Lecithin TCI (Shanghai) Chemical Industry Development Co., Ltd. L0023 cholesterol Bailingwei Technology Co., Ltd. 156754 Twain-80 Shanghai MacLean Biochemical Technology Co., Ltd. T818928 Anhydrous ethanol Beijing Myrida Technology Co., Ltd. M042753 Chloroform Modern Oriental (Beijing) Technology Development Co., Ltd. 67-66-3 Astaxanthin powder (pure) Nanjing Dulai Biotechnology Co., Ltd. V0041

[0056] Example 1 A method for preparing nano-astaxanthin liposomes

[0057] The following steps are involved:

[0058] Accurately weigh 250 mg of lecithin, 50 mg of cholesterol, and 40 mg of Haematococcus pluvialis extract, and fully dissolve them in 100 mL of anhydrous ethanol; transfer them to a rotary evaporator and evaporate them in a 35°C water bath on a rotary evaporator. Vacuum condensation and rotary evaporation are used to remove the organic solvent, and a uniform lipid film containing astaxanthin is formed at the bottom of the rotary evaporator. Subsequently, 50 mL of phosphate buffered saline solution and 10 mL of Tween-80 are added to hydrate and dissolve, and then ultrasonic cell crusher is used for ultrasonication to obtain the nano-astaxanthin liposomes.

[0059] Example 2

[0060] The only difference from Example 1 is that the amount of Tween-80 is 1.6 ml, and the other steps are the same as Example 1.

[0061] Example 3

[0062] The only difference from Example 1 is that the amount of Tween-80 is 5 ml, and the other steps are the same as those in Example 1.

[0063] Example 4

[0064] The only difference from Example 1 is that the amount of Tween-80 is 15 ml, and the other steps are the same as those in Example 1.

[0065] Example 5

[0066] The only difference from Example 1 is that anhydrous ethanol is replaced by chloroform, and the other steps are the same as those in Example 1.

[0067] Example 6

[0068] The only difference from Example 1 is that the phosphate buffered saline solution is replaced with pure water, and the other steps are the same as those in Example 1.

[0069] Example 7 A method for preparing nano-astaxanthin liposomes

[0070] The following steps are involved:

[0071] Accurately weigh 50 mg of lecithin, 10 mg of cholesterol, and 20 mg of Haematococcus pluvialis extract, and fully dissolve them in 30 mL of anhydrous ethanol; transfer them to a rotary evaporator and evaporate them in a 35°C water bath. Vacuum condensation and rotary evaporation are used to remove the organic solvent, and a uniform lipid film containing astaxanthin is formed at the bottom of the rotary evaporator. Subsequently, 20 mL of phosphate buffered saline solution and 10 mL of Tween-80 are added to hydrate and dissolve, and then ultrasonic cell crusher is used for ultrasonication to obtain the nano-astaxanthin liposomes.

[0072] Example 8 A method for preparing nano-astaxanthin liposomes

[0073] The following steps are involved:

[0074] Accurately weigh 150 mg of lecithin, 30 mg of cholesterol, and 30 mg of Haematococcus pluvialis extract, and fully dissolve them in 60 mL of anhydrous ethanol; transfer them to a rotary evaporator and evaporate them in a 40°C water bath on a rotary evaporator. Vacuum condensation and rotary evaporation are used to remove the organic solvent, and a uniform lipid film containing astaxanthin is formed at the bottom of the rotary evaporator. Subsequently, 30 mL of phosphate buffered saline solution and 10 mL of Tween-80 are added to hydrate and dissolve, and then ultrasonic cell crusher is used for ultrasonication to obtain the nano-astaxanthin liposomes.

[0075] Comparative Example 1

[0076] According to the method described in the reference document CN110558435A, a nano-astaxanthin liposome was prepared. The specific preparation method is as follows:

[0077] Accurately weigh 250 mg of lecithin, 50 mg of cholesterol, and 40 mg of Haematococcus pluvialis extract, and fully dissolve them in 100 mL of anhydrous ethanol; transfer them to a rotary evaporator and evaporate them in a 35°C water bath. Vacuum condensation and rotary evaporation are used to remove the organic solvent, and a uniform lipid film containing astaxanthin is formed at the bottom of the rotary evaporator. Subsequently, 50 mL of phosphate buffered saline solution is added to hydrate and dissolve, and then ultrasonic cell crusher is used for ultrasonication to obtain the nano-astaxanthin liposomes.

[0078] Comparative Example 2: A method for preparing nano-astaxanthin liposomes

[0079] The following steps are involved:

[0080] Accurately weigh 250 mg of lecithin, 50 mg of cholesterol, and 4 mg of astaxanthin powder (pure product), and fully dissolve them in 100 mL of anhydrous ethanol; transfer them to a rotary evaporator and evaporate them in a 35°C water bath. Vacuum condensation and rotary evaporation are used to remove the organic solvent, and a uniform lipid film containing astaxanthin is formed at the bottom of the rotary evaporator. Subsequently, 50 mL of phosphate buffered saline solution and 10 mL of Tween-80 are added to hydrate and dissolve, and then ultrasonic cell crusher is used for ultrasonication to obtain the nano-astaxanthin liposomes.

[0081] Comparative Example 3

[0082] According to the method described in the reference document CN117017802A, a nano-astaxanthin liposome was prepared. The specific preparation method is as follows:

[0083] 10 mL of Tween-80, 250 mg of lecithin, 50 mg of cholesterol, and 40 mg of Haematococcus pluvialis extract were dissolved in anhydrous ethanol, then transferred to a rotary evaporator, and rotary evaporated in a 35°C water bath on a rotary evaporator. The organic solvent was removed by vacuum condensation and rotary evaporation, and a uniform lipid film containing astaxanthin was formed at the bottom of the rotary evaporator. Subsequently, 50 mL of phosphate buffered saline solution was added for hydration, and then ultrasonic cell crusher was used for ultrasonication to obtain the nano-astaxanthin liposomes.

[0084] Comparative Example 4

[0085] Accurately weigh 25 mg of lecithin, 3 mg of cholesterol, and 10 mg of Haematococcus pluvialis extract, and fully dissolve them in 10 mL of anhydrous ethanol; transfer them to a rotary evaporator and evaporate them in a 35°C water bath on a rotary evaporator. Vacuum condensation and rotary evaporation are used to remove the organic solvent, and a uniform lipid film containing astaxanthin is formed at the bottom of the rotary evaporator. Subsequently, 10 mL of phosphate buffered saline solution is added to hydrate and dissolve, and then ultrasonic cell crusher is used for ultrasonication to obtain the nano-astaxanthin liposomes.

[0086] Application Example 1 A kind of essence water

[0087] Its formula components are:

[0088]

[0089] The preparation method is as follows: the nano-astaxanthin liposomes and the above-mentioned essence water component are mixed, stirred evenly, and respectively packaged into the same container.

[0090] Application Example 2 A facial cream

[0091] Its formula components are:

[0092]

[0093] The preparation method is as follows: the nano-astaxanthin liposomes and the above-mentioned face cream components are mixed, stirred evenly, and respectively packaged into the same container.

[0094] Effect Experiment

[0095] 1. Determination of Nano-astaxanthin Liposome Encapsulation Efficiency

[0096] The following steps are involved:

[0097] (1) Standard solution used to prepare the astaxanthin standard curve:

[0098] Prepare 800 μg / mL astaxanthin solution: dissolve 8 mg astaxanthin powder (pure product) in 10 mL anhydrous ethanol and store in a -20°C refrigerator.

[0099] Prepare 8μg / mL astaxanthin solution: Take 0.1mL of 800μg / mL astaxanthin solution and dilute to 10mL with chloroform. Take an appropriate amount of solution and scan the wavelength in the range of 200-800nm ​​using a UV spectrophotometer to determine that the wavelength of the maximum absorption peak of astaxanthin is 490nm.

[0100] Take 3mL of 8μg / mL astaxanthin solution and dilute it to 6mL with chloroform to obtain an astaxanthin solution with a concentration of 4μg / mL, and then dilute it with chloroform in a concentration gradient. The concentration series of astaxanthin solutions were 4μg / mL, 2μg / mL, 1μg / mL, 0.5μg / mL, and 0.25μg / mL, respectively. Measure at the wavelength of the maximum absorption peak of astaxanthin and draw an astaxanthin-chloroform standard curve. The results are as follows: Figure 1 , Figure 1 UV absorption images of astaxanthin solutions with different concentrations.

[0101] (2) Draw a standard curve for measuring astaxanthin encapsulation efficiency:

[0102] The ordinate is the absorbance obtained by the UV spectrophotometer, and the abscissa is the astaxanthin concentration. The best standard curve for detecting the astaxanthin encapsulation efficiency is obtained by fitting. The results are shown in Figure 2 , Figure 2 It is a standard curve for detecting the astaxanthin encapsulation efficiency. The standard curve equation has the form shown in formula (II):

[0103] y=0.15441x+0.00654

[0104] Where y is the absorbance detected by UV spectrophotometer, x is the astaxanthin concentration, R 2 =0.999.

[0105] (3) Determination of nano-astaxanthin liposome encapsulation efficiency:

[0106] The encapsulation rate of nano-astaxanthin liposomes was determined by petroleum ether extraction. Take 1 mL of sample solution, add 10 mL of petroleum ether, fully shake and stir at 30°C for 5 minutes (ultrasound or shake well), let it stand for 30 minutes, transfer the upper liquid to a rotary evaporator, and add 10 mL of petroleum ether for extraction, and repeat twice. Vacuum the upper liquid at 50°C to remove the petroleum ether and precipitate the free astaxanthin. Add 2 mL of chloroform to redissolve it and measure its absorbance at a wavelength of 490 nm. Calculate the content of free astaxanthin based on the standard curve. The encapsulation rate has the form shown in formula (III):

[0107]

[0108] Table 1 Encapsulation efficiency of each test sample

[0109] Test samples Encapsulation rate (%) Example 1 99.51 Example 2 97.41 Example 3 97.00 Example 4 98.73 Example 5 98.35 Example 6 98.99 Example 7 97.31 Example 8 96.11 Comparative Example 1 82.82 Comparative Example 2 88.65 Comparative Example 3 83.29 Comparative Example 4 90.76

[0110] According to the experimental data of Table 1, it can be seen that the encapsulation efficiency of the nano-astaxanthin liposomes prepared by Examples 1-8 is higher than that of Comparative Examples 1-4, and the encapsulation efficiency of the nano-astaxanthin liposomes prepared by Example 1 can reach 99%. Wherein, when preparing nano-astaxanthin liposomes, Examples 1-8 add Tween-80 after forming the astaxanthin liposome film, so the encapsulation efficiency of the nano-astaxanthin liposomes prepared by Examples 1-8 is significantly improved; Comparative Example 1 does not add Tween-80, and Comparative Example 3 adds Tween-80 before forming the astaxanthin liposome film when preparing nano-astaxanthin liposomes, and the effect is not ideal. This is because the astaxanthin liposome film has a certain wall hanging phenomenon after hydration ultrasound, and the loss rate is large, so in order to reduce the loss rate of the system, Tween-80 needs to be added to improve solubility. It can be seen that the order of adding Tween-80 has a great influence on the encapsulation efficiency of nano-astaxanthin liposomes. The ratios of the Haematococcus pluvialis extract, cholesterol and lecithin used in Comparative Example 4 are not within the scope of not applying for protection, and the encapsulation efficiency of the nano-astaxanthin liposomes prepared by Comparative Example 4 is also not ideal. In addition, compared with Comparative Example 2, the encapsulation efficiency of Example 1 is significantly improved, which shows that the higher the astaxanthin content, the better.

[0111] 2. The determination of the particle size of nano-astaxanthin liposomes comprises the following steps:

[0112] Take 0.5 mL of the prepared nano-astaxanthin liposome sample solution, add 1.5 mL of ultrapure water and mix thoroughly, place in a polystyrene cuvette, and use the Nano-ZS90 laser particle size analyzer from Malvern to measure the particle size and particle size distribution of the nano-astaxanthin liposome.

[0113] Table 2 Particle size of each test sample

[0114] Test samples Particle size (nm) PDI Example 1 76.70 0.2854 Example 2 88.28 0.409 Example 3 85.00 0.3881 Example 4 91.09 0.513 Example 5 83.16 0.3032 Example 6 86.06 0.4285 Example 7 81.67 0.4106 Example 8 95.17 0.509 Comparative Example 1 140.86 0.6642 Comparative Example 2 100.50 0.5878 Comparative Example 3 105.92 0.5956 Comparative Example 4 95.90 0.5078

[0115] According to the experimental data in Table 2, it can be seen that the particle size of the nano-astaxanthin liposomes prepared in Examples 1-8 is smaller than that in Comparative Examples 1-4. Among them, the nano-astaxanthin liposomes prepared in Example 1 have the smallest particle size, the most uniform dispersion, and the most stable system. Comparative Example 4 adjusts the ratio of the Haematococcus pluvialis extract to cholesterol and lecithin. Although the particle size of the prepared nano-astaxanthin liposomes is not much different from that of Example 8, it can be seen from Table 1 that the encapsulation efficiency of Comparative Example 4 is not as good as that of Example 8.

[0116] 3. The determination of the DPPH free radical scavenging rate of nano-astaxanthin liposomes comprises the following steps:

[0117] The DPPH free radical scavenging ability of astaxanthin liposomes was determined by spectrophotometry. The characteristic absorption peak of DPPH-anhydrous ethanol solution with purple color at 517nm was used. After adding antioxidants, the decrease of the absorbance at 517nm indicated its scavenging ability for DPPH free radicals.

[0118] (1) Preparation of DPPH ethanol solution: Weigh 1 mg DPPH and add 12.5 mL anhydrous ethanol to dissolve it. The DPPH concentration is prepared to 2 × 10 -4 mol / L; stored at 0-4℃ away from light; vitamin C (0.5mg / mL) was used as the positive control.

[0119] (2) Preparation of test solution: The sample was prepared into 4 concentrations, diluted 0 times, 5 times, 10 times and 20 times respectively.

[0120] (3) Take 1 mL of the test solution and 1 mL of 2×10 -4 mol / L DPPH solution mixed (A);

[0121] (4) Take 1 mL of anhydrous ethanol and 1 mL of 2×10 -4 mol / L DPPH solution mixed (B);

[0122] (5) Mix 1 mL of anhydrous ethanol with 1 mL of the test solution (C);

[0123] (6) After 30 minutes of reaction, measure the absorbance values ​​of A, B, and C at 517 nm. The scavenging rate K of the sample for DPPH can be calculated using the following formula (IV):

[0124]

[0125] Among them, A is the absorbance of the mixed solution of DPPH and nano-astaxanthin liposomes; B is the absorbance of the mixed solution of DPPH and anhydrous ethanol; C is the absorbance of the mixed solution of nano-astaxanthin liposomes and anhydrous ethanol.

[0126] Table 3 DPPH removal rate of each test sample

[0127] Test samples Clearance rate (%) Example 1 80.94±8.38 Example 2 70.51±2.06 Example 3 72.25±5.73 Example 4 75.03±9.44 Example 5 75.89±4.15 Example 6 77.68±4.48 Example 7 68.52±2.88 Example 8 73.10±8.34 Comparative Example 1 54.97±5.53 Comparative Example 2 64.51±1.83 Comparative Example 3 60.35±3.68 Comparative Example 4 65.27±2.45

[0128] According to the experimental data in Table 3, it can be seen that the nano-astaxanthin liposomes prepared in Examples 1-8 have a better effect on the removal of DPPH, and the removal rate of DPPH in Example 1 can reach 80.94±8.38%. The removal rate of DPPH by the nano-astaxanthin liposomes prepared in Comparative Examples 1-4 is much lower than that in Example 1. Although the nano-astaxanthin liposomes prepared in Example 1 have a similar DPPH removal rate to that in Example 5, chloroform is toxic and cannot be used in cosmetics. It can be seen from Examples 1-4 that different addition amounts of Tween-80 have a great influence on the DPPH removal rate. When the addition amount of Tween-80 is 10mL, the DPPH removal rate of the nano-astaxanthin liposomes is much higher than that of the nano-astaxanthin liposomes when the addition amount is 1.6mL, 5mL and 15mL Tween-80.

[0129] Taking all factors into consideration, the effect of the nano-astaxanthin liposomes prepared using the components and methods disclosed in this application is optimal.

[0130] 4. The stability test of nano-astaxanthin liposomes in the field of cosmetics may specifically include the following steps:

[0131] Nano-astaxanthin liposome stability test: Attention should be paid to items that are easy to change during storage and affect quality and safety, and the law of changes in the properties of nano-astaxanthin liposomes (color, odor, etc.) under the influence of temperature, light, etc. over time should be observed.

[0132] Formula stability test: Attention should be paid to whether there are chemical and / or biological interactions between the raw materials of the formula (essence water, cream), and whether stratification occurs in the formula over time.

[0133] The essence water and cream prepared in Application Example 1 and Application Example 2 were placed under 6 conditions of high temperature, freezing, alternating hot and cold, light, room temperature, and refrigeration for 7 days, and photographed and recorded on the 1st, 3rd, 5th, and 7th days to see whether the nano astaxanthin liposomes or the formula changed color, faded, or stratified over time.

[0134] A High temperature test: Place the samples (nano-astaxanthin liposomes, essence water, and facial cream) in a sealed clean container, and then place it in a constant temperature incubator pre-adjusted to 50°C for 7 days. Take samples on the 1st, 3rd, 5th, and 7th days, and take photos after returning to room temperature.

[0135] B Freezing test: Place the sample in a -20℃ refrigerator for 7 days, take samples on the 1st, 3rd, 5th and 7th days, and take photos after returning to room temperature.

[0136] C Hot and cold alternating test: Store the sample in a sealed clean container, then place it in a refrigerator pre-adjusted to -20℃, take it out after 24 hours, move it to a constant temperature incubator pre-adjusted to 50℃, place it in an incubator for 24 hours, take it out, and alternate between the two at intervals of 24 hours. Repeat this cycle for 7 days, and observe after returning to room temperature.

[0137] D. Light irradiation test: Take an appropriate amount of sample and place it in a suitable light-transmitting container under a transparent glass window. Take samples and observe them on the 1st, 3rd, 5th and 7th day. During the test, pay attention to controlling the temperature to keep it consistent with the room temperature, and pay attention to observing the changes in the appearance of the sample.

[0138] E Normal temperature test: Place the sample in a sealed clean container in a dark and dry place for 7 days. Take samples on the 1st, 3rd, 5th and 7th day and take photos for observation.

[0139] F Refrigeration test: Place the sample in a refrigerator at 4°C for 7 days, take samples on the 1st, 3rd, 5th and 7th days, and take photos after returning to room temperature.

[0140] The results show that the nano-astaxanthin liposomes showed almost no discoloration under all conditions ( Figure 7 ); In the formula, the essence water fades slightly under light conditions, and the 5% and 10% essence water fades slightly under high temperature conditions ( Figure 8 ); The cream fades slightly under light conditions, and the 5% and 10% creams fade slightly under high temperature conditions ( Fig. 9 ).

[0141] The above results indicate that the nano-astaxanthin liposomes prepared in a specific ratio according to the present invention have the advantages of simple preparation method, safety and non-toxicity, good encapsulation rate, high stability, outstanding antioxidant activity, uniform particle size, good dispersibility, and significantly improved water solubility, and can be applied in the field of cosmetics, greatly improving the bioavailability of astaxanthin.

[0142] The above description is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several modifications or equivalent substitutions can be made to the technical solution without departing from the principle of the present invention. These modifications or equivalent substitutions should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing nano-astaxanthin liposomes, characterized in that: The following steps are involved: (1) dissolving a Haematococcus pluvialis extract, lecithin and cholesterol in an organic solvent and reacting them under heating conditions to obtain a lipid film containing astaxanthin; (2) adding a hydrating solvent and Tween-80 to the lipid film obtained in step (1), and subjecting the mixture to ultrasound to obtain the nano-astaxanthin liposomes.

2. The preparation method according to claim 1, characterized in that: The mass ratio of the Haematococcus pluvialis extract, lecithin and cholesterol is 20-50:50-250:10-50.

3. The preparation method according to claim 2, characterized in that: The mass ratio of the Haematococcus pluvialis extract, lecithin and cholesterol is 40:250:

50.

4. The preparation method according to claim 1, characterized in that: The heating temperature is 35°C-40°C.

5. The preparation method according to claim 1, characterized in that: The organic solvent is anhydrous ethanol and / or chloroform.

6. The preparation method according to claim 1, characterized in that: The hydration solvent is phosphate buffer or pure water.

7. The preparation method according to claim 1, characterized in that: The volume ratio of the total mass of the Haematococcus pluvialis extract, lecithin and cholesterol described in step (1) to the hydration solvent and Tween-80 described in step (2) is 80-340 mg:10-50 mL:1-15 mL.

8. The preparation method according to claim 7, characterized in that: The volume ratio of the total mass of the Haematococcus pluvialis extract, lecithin and cholesterol described in step (1) to the hydration solvent and Tween-80 described in step (2) is 340 mg:50 mL:15 mL.

9. A nano-astaxanthin liposome prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the nano-astaxanthin liposomes prepared by the preparation method according to any one of claims 1 to 8 in the preparation of cosmetics.

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