A nano astaxanthin liposome, a preparation method and application thereof

Abstract

The application belongs to the technical field of cosmetics, and discloses a kind of nano astaxanthin liposome and its preparation method and application.The preparation method of the nano astaxanthin liposome includes the following steps: the extract of haematococcus pluvialis, lecithin and cholesterol are dissolved in an organic solvent, heated to 35-40 DEG C for reaction, vacuum condensation is removed to obtain uniform astaxanthin-containing lipid film;phosphate buffer solution and tween-80 are then added to the solution, and then ultrasonic is performed to obtain nano astaxanthin liposome.The nano astaxanthin liposome has uniform particle size, good dispersibility, high encapsulation efficiency and stability, outstanding antioxidant activity, simple preparation method, and is safe and non-toxic, which can be applied to the preparation of cosmetics and large-scale production.

Description

Technical Field

[0001] This invention relates to the field of cosmetics, and more specifically, to a nano-astaxanthin liposome, a method for preparing the nano-astaxanthin liposome, and its application. Background Technology

[0002]

[0003] Astaxanthin (ASX) is a red, fat-soluble carotenoid, belonging to the xanthophyll family along with other carotenoids such as β-cryptoxanthin, β-carotene, lycopene, and zeaxanthin. It is produced by various microorganisms and marine animals. The central nonpolar region of astaxanthin consists of 13 bonds, giving it the ability to remove high-energy electrons. The hydroxyl and ketone groups on the two rings increase its polarity, significantly enhancing its ability to cross cell membranes. These unique chemical properties endow astaxanthin with several 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, insoluble in water, and easily decomposes in light, exhibiting poor stability, which greatly limits its application in cosmetics. Therefore, developing a carrier system that can improve its solubility and stability is imperative.

[0004] Liposomes are closed microvesicles with a biomembrane-like structure formed from phospholipids. They possess both hydrophilic and hydrophobic properties, enabling them to encapsulate both water-soluble and lipid-soluble substances. The membrane material for liposomes is generally made from natural raw materials, offering advantages in biocompatibility and safety. Liposome encapsulation can increase the solubility of astaxanthin in aqueous solutions. Astaxanthin can penetrate the liposome membrane and interact with the membrane's polar groups through hydrogen bonds. Therefore, using liposome carrier technology to prepare astaxanthin liposomes can solve the water solubility problem of astaxanthin and improve its effective utilization rate.

[0005] For example, Chinese patent CN110558435A discloses an astaxanthin nanoliposome, its preparation method, and its application. This preparation method involves dissolving astaxanthin, cholesterol, and phospholipids in an organic solvent, removing the organic solvent by vacuum evaporation to form a thin film, and then adding a buffer solution to dissolve and disperse the film. This invention exhibits excellent characterization properties, a high synthesis rate, and a simple process. However, the film formed during synthesis suffers from some wall adhesion, resulting in a relatively high loss rate and thus reducing the utilization rate of astaxanthin.

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

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

[0008] To address the problems existing in current technologies, this invention aims to provide astaxanthin liposomes, their preparation method, and applications. The key raw material used is astaxanthin, derived from natural Haematococcus pluvialis extract, which is recognized worldwide as the richest natural source of astaxanthin. The nano-astaxanthin liposomes prepared by the method of this invention can improve the solubility, stability, and antioxidant activity of astaxanthin. Furthermore, the process is simple, the encapsulation effect is easy to detect, it is safe and non-toxic, and the bioavailability of astaxanthin is improved.

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

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

[0011] (1) Haematococcus pluvialis extract, lecithin and cholesterol were dissolved in an organic solvent and reacted under heating conditions to obtain a lipid film containing astaxanthin.

[0012] (2) Add hydration solvent and Tween-80 to the lipid film obtained in step (1) and sonicate to obtain the nano-astaxanthin liposomes.

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

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

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

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

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

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

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

[0020] Add the organic solvent until all the Haematococcus pluvialis extract, lecithin, and cholesterol are completely dissolved.

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

[0022] The hydration solvent mentioned 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 a phosphate buffer solution.

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

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

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

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

[0028] Preferably, the specific steps of the reaction in step (1) are as follows: Haematococcus pluvialis extract, lecithin and cholesterol are dissolved in an organic solvent and mixed evenly, heated to 35℃-40℃ for rotary evaporation, and the organic solvent is removed by vacuum condensation to obtain a uniform lipid film containing astaxanthin.

[0029] During the implementation of this invention, it was discovered that even after hydrating and ultrasonicating astaxanthin-containing lipid films with only an aqueous solvent, a certain degree of adhesion to the film still occurred. Therefore, this invention added Tween-80 to the aqueous solvent. By controlling the volume ratio of the aqueous solvent to Tween-80, the solubility of the astaxanthin lipid film can be significantly improved, thereby eliminating the adhesion phenomenon.

[0030] A preferred embodiment is as follows: 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 flask, heated to 35°C-40°C for rotary evaporation, and the organic solvent is removed by vacuum condensation to obtain a uniform lipid film containing astaxanthin. Subsequently, 50 mL of phosphate buffer solution and 10 mL of Tween-80 are added for hydration and dissolution, followed by ultrasonication using an ultrasonic cell disruptor to obtain the astaxanthin liposomes.

[0031] The present invention also provides a nano-astaxanthin liposome prepared by the above method.

[0032] This invention also provides the application of the nano-astaxanthin liposomes obtained by the above preparation method in the field of cosmetics.

[0033] Studies have shown that the stability of the nano-astaxanthin lipids prepared in this application is greatly improved under different conditions, and they are safe and non-toxic to the human body, making them more suitable for use 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 rate, uniform particle size, and good dispersibility.

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

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

[0039] (5) The preparation process of the nano-astaxanthin liposomes is simple and suitable for large-scale production. Attached Figure Description

[0040] Figure 1 Ultraviolet absorption images of astaxanthin solutions at different concentrations;

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

[0042] Figure 3 Images showing the particle size data of nano-astaxanthin liposomes prepared in Examples 1-8;

[0043] Illustrations: A represents Example 1; B represents Example 2; C represents Example 3; D represents Example 4; E represents Example 5; F represents Example 6; G represents Example 7; H represents Example 8;

[0044] Figure 4 Images showing the particle size data of nano-astaxanthin liposomes prepared in comparative examples 1-4;

[0045] Illustrations: a is Comparative Example 1; b is Comparative Example 2; c is Comparative Example 3; d is Comparative Example 4;

[0046] Figure 5 Images showing the DPPH free radical scavenging rate of the nano-astaxanthin liposomes prepared in Examples 1-8;

[0047] Illustrations: A represents Example 1; B represents Example 2; C represents Example 3; D represents Example 4; E represents Example 5; F represents Example 6; G represents Example 7; H represents Example 8;

[0048] Figure 6 Images showing the DPPH free radical scavenging rate of nano-astaxanthin lipids prepared in comparative examples 1-4;

[0049] Illustrations: a is Comparative Example 1; b is Comparative Example 2; c is Comparative Example 3; d is Comparative Example 4;

[0050] Figure 7 Photographs showing the stability of nano-astaxanthin liposomes under different storage conditions;

[0051] Figure 8 Photos showing the stability of astaxanthin essence water under different storage conditions;

[0052] Figure 9 Photos showing the stability of astaxanthin face cream under different storage conditions. Detailed Implementation

[0053] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the materials and reagents used are commercially available.

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

[0055] reagents factory Item number Haematococcus pluvialis extract Shanxi Zizhe Biotechnology 472-61-7 Lecithin THIAI (Shanghai) Chemical Industry Development Co., Ltd. L0023 cholesterol Bailingwei Technology Co., Ltd. 156754 Twain-80 Shanghai McLean Biochemical Technology Co., Ltd. T818928 Anhydrous ethanol Beijing Mairuida 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] Includes the following steps:

[0058] Accurately weigh 250 mg of lecithin, 50 mg of cholesterol, and 40 mg of Haematococcus pluvialis extract, and dissolve them thoroughly in 100 mL of anhydrous ethanol. Transfer the solution to a rotary evaporator flask and evaporate it in a 35°C water bath. Remove the organic solvent by vacuum condensation and rotary evaporation, forming a uniform lipid film containing astaxanthin at the bottom of the flask. Then add 50 mL of phosphate buffer solution and 10 mL of Tween-80 hydration solution to dissolve the astaxanthin. Finally, sonicate the solution using an ultrasonic cell disruptor 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 in 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 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 in Example 1.

[0065] Example 5

[0066] The only difference from Example 1 is that anhydrous ethanol is replaced with chloroform; the other steps are the same as in Example 1.

[0067] Example 6

[0068] The only difference from Example 1 is that the phosphate buffer solution is replaced with pure water; the other steps are the same as in Example 1.

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

[0070] Includes the following steps:

[0071] Accurately weigh 50 mg of lecithin, 10 mg of cholesterol, and 20 mg of Haematococcus pluvialis extract, and dissolve them thoroughly in 30 mL of anhydrous ethanol. Transfer the solution to a rotary evaporator flask and evaporate it in a 35°C water bath. Remove the organic solvent by vacuum condensation and rotary evaporation, forming a uniform lipid film containing astaxanthin at the bottom of the flask. Then add 20 mL of phosphate buffer solution and 10 mL of Tween-80 hydration solution to dissolve the astaxanthin. Finally, sonicate the solution using an ultrasonic cell disruptor to obtain the nano-astaxanthin liposomes.

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

[0073] Includes the following steps:

[0074] Accurately weigh 150 mg of lecithin, 30 mg of cholesterol, and 30 mg of Haematococcus pluvialis extract, and dissolve them thoroughly in 60 mL of anhydrous ethanol. Transfer the solution to a rotary evaporator flask and evaporate it in a 40°C water bath. Remove the organic solvent by vacuum condensation and rotary evaporation, forming a uniform lipid film containing astaxanthin at the bottom of the flask. Then add 30 mL of phosphate buffer solution and 10 mL of Tween-80 hydration solution to dissolve the astaxanthin. Finally, sonicate the solution using an ultrasonic cell disruptor to obtain the nano-astaxanthin liposomes.

[0075] Comparative Example 1

[0076] A nano-astaxanthin liposome was prepared according to the method described in prior art CN110558435A. 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 dissolve them thoroughly in 100 mL of anhydrous ethanol. Transfer the solution to a rotary evaporator flask and evaporate it in a 35°C water bath. Remove the organic solvent by vacuum condensation and rotary evaporation, forming a uniform lipid film containing astaxanthin at the bottom of the flask. Then, add 50 mL of phosphate buffer solution to hydrate and dissolve the astaxanthin, followed by ultrasonication using an ultrasonic cell disruptor to obtain the nano-astaxanthin liposomes.

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

[0079] Includes the following steps:

[0080] Accurately weigh 250 mg of lecithin, 50 mg of cholesterol, and 4 mg of astaxanthin powder (pure), and dissolve them thoroughly in 100 mL of anhydrous ethanol. Transfer the solution to a rotary evaporator flask and evaporate it in a 35°C water bath. Remove the organic solvent by vacuum condensation and rotary evaporation, forming a uniform lipid film containing astaxanthin at the bottom of the flask. Then add 50 mL of phosphate buffer solution and 10 mL of Tween-80 hydration solution to dissolve the astaxanthin. Finally, sonicate the solution using an ultrasonic cell disruptor to obtain the nano-astaxanthin liposomes.

[0081] Comparative Example 3

[0082] A nano-astaxanthin liposome was prepared according to the method described in prior art CN117017802A. 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 and then transferred to a rotary evaporator flask. The mixture was then rotary evaporated at 35°C in a water bath. The organic solvent was removed by vacuum condensation and rotary evaporation, forming a uniform lipid film containing astaxanthin at the bottom of the flask. Subsequently, 50 mL of phosphate buffer solution was added for hydration, followed by ultrasonication using an ultrasonic cell disruptor 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 dissolve them thoroughly in 10 mL of anhydrous ethanol. Transfer the solution to a rotary evaporator flask and evaporate it in a 35°C water bath. Remove the organic solvent by vacuum condensation and rotary evaporation, forming a uniform lipid film containing astaxanthin at the bottom of the flask. Then, add 10 mL of phosphate buffer solution to hydrate and dissolve the astaxanthin, and finally sonicate using an ultrasonic cell disruptor to obtain the nano-astaxanthin liposomes.

[0086] Application Example 1: An Essence Water

[0087] Its formula components are:

[0088]

[0089] The preparation method is as follows: mix the nano-astaxanthin liposomes and the above-mentioned essence water components, stir evenly, and then dispense them into the same containers.

[0090] Application Example 2: A face cream

[0091] Its formula components are:

[0092]

[0093] The preparation method is as follows: mix the nano-astaxanthin liposomes and the above-mentioned face cream components, stir evenly, and then dispense them into the same containers.

[0094] Effect Experiment

[0095] 1. Determination of encapsulation efficiency of nano-astaxanthin liposomes

[0096] Includes the following steps:

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

[0098] To prepare an 800 μg / mL astaxanthin solution: Dissolve 8 mg of astaxanthin powder (pure product) in 10 mL of anhydrous ethanol and store at -20°C.

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

[0100] 3 mL of 8 μg / mL astaxanthin solution was diluted to 6 mL with chloroform to obtain an astaxanthin solution with a concentration of 4 μg / mL. This solution was then serially diluted with chloroform to obtain astaxanthin solutions with concentrations of 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, and 0.25 μg / mL. Measurements were taken at the wavelength of the maximum absorption peak of astaxanthin, and an astaxanthin-chloroform standard curve was plotted. The results are as follows: Figure 1 , Figure 1 The images show the UV absorption of astaxanthin solutions of different concentrations.

[0101] (2) Plot the standard curve used to measure the encapsulation efficiency of astaxanthin:

[0102] The vertical axis represents the absorbance obtained by a UV spectrophotometer, and the horizontal axis represents the astaxanthin concentration. A standard curve optimal for detecting astaxanthin encapsulation efficiency was obtained through fitting, and the results are as follows: Figure 2 , Figure 2 A standard curve for detecting the encapsulation efficiency of astaxanthin. The equation for this standard curve has the form shown in equation (II):

[0103] y = 0.15441x + 0.00654

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

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

[0106] The encapsulation efficiency of nano-astaxanthin liposomes was determined using petroleum ether extraction. 1 mL of sample solution was added to 10 mL of petroleum ether, and the mixture was thoroughly shaken and stirred at 30°C for 5 min (ultrasonic or shaken). After standing for 30 min, the supernatant was transferred to a rotary evaporator flask, and another 10 mL of petroleum ether was added for extraction. This process was repeated twice. The supernatant was then vacuum-evaporated at 50°C to remove the petroleum ether, allowing free astaxanthin to precipitate. 2 mL of chloroform was added to redissolve the astaxanthin, and the absorbance was measured at 490 nm. The content of free astaxanthin was calculated based on the standard curve. The encapsulation efficiency has the form shown in formula (III):

[0107]

[0108] Table 1 Encapsulation efficiency of each test sample

[0109] Test sample Encapsulation efficiency (%) 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] As shown in Table 1, the encapsulation efficiency of the nano-astaxanthin liposomes prepared in Examples 1-8 was higher than that in Comparative Examples 1-4, with Example 1 achieving an encapsulation efficiency of 99%. Specifically, in Examples 1-8, Tween-80 was added after the formation of the astaxanthin liposome film, resulting in a significantly improved encapsulation efficiency. Comparative Example 1 did not add Tween-80, and Comparative Example 3 added Tween-80 before the formation of the astaxanthin liposome film, with unsatisfactory results. This is because the astaxanthin liposome film exhibits some wall adhesion after hydration and ultrasound, resulting in a relatively high loss rate. Therefore, Tween-80 is added to improve solubility and reduce the loss rate. Thus, the order of Tween-80 addition significantly affects the encapsulation efficiency of the nano-astaxanthin liposomes. The proportions of Haematococcus pluvialis extract, cholesterol, and lecithin used in Comparative Example 4 are all outside the scope of protection sought. Furthermore, the encapsulation efficiency of the nano-astaxanthin liposomes prepared from Comparative Example 4 is also unsatisfactory. In addition, Example 1 showed a significantly improved encapsulation efficiency compared to Comparative Example 2, indicating that a higher astaxanthin content is not necessarily better.

[0111] 2. The determination of the particle size of nano-astaxanthin liposomes includes 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 the mixture in a polystyrene cuvette and use a Malvern Nano-ZS90 laser particle size analyzer to determine the particle size and particle size distribution of the nano-astaxanthin liposomes.

[0113] Table 2 Particle size of each test sample

[0114] Test sample 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] As can be seen from the experimental data in Table 2, 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 adjusted the ratio of Haematococcus pluvialis extract to cholesterol and lecithin. Although the particle size of the prepared nano-astaxanthin liposomes was not significantly different from that in Example 8, Table 1 shows that the encapsulation efficiency of Comparative Example 4 was lower than that of Example 8.

[0116] 3. The determination of the DPPH free radical scavenging rate of nano-astaxanthin liposomes includes 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 at 517 nm (a purple cluster) was utilized; after adding an antioxidant, the decrease in absorbance at 517 nm was used to represent its DPPH free radical scavenging ability.

[0118] (1) Preparation of DPPH ethanol solution: Weigh 1 mg DPPH, add 12.5 mL of anhydrous ethanol to dissolve it, and prepare a DPPH concentration of 2 × 10⁻⁶. -4 mol / L; store at 0-4℃ protected from light; use vitamin C (0.5 mg / mL) as a positive control.

[0119] (2) Preparation of test solution: The sample was prepared into 4 concentrations and 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 Mix the mol / L DPPH solution thoroughly (A);

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

[0122] (5) Take 1 mL of anhydrous ethanol and mix it with 1 mL of the test solution (C);

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

[0124]

[0125] Where A is the absorbance of the DPPH and nano-astaxanthin liposome mixed solution; B is the absorbance of the DPPH and anhydrous ethanol mixed solution; and C is the absorbance of the nano-astaxanthin liposome and anhydrous ethanol mixed solution.

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

[0127] Test sample 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] As shown in Table 3, the nano-astaxanthin liposomes prepared in Examples 1-8 exhibit better DPPH scavenging effects, with Example 1 achieving a DPPH scavenging rate of 80.94 ± 8.38%. The nano-astaxanthin liposomes prepared in Comparative Examples 1-4 show significantly lower DPPH scavenging rates than Example 1. Although the DPPH scavenging rates of the nano-astaxanthin liposomes prepared in Example 1 are not significantly different from those in Example 5, chloroform is toxic and cannot be used in cosmetics. Examples 1-4 demonstrate that different amounts of Tween-80 significantly affect the DPPH scavenging rate. The nano-astaxanthin liposomes with 10 mL of Tween-80 exhibit a much higher DPPH scavenging rate than those with 1.6 mL, 5 mL, and 15 mL of Tween-80.

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

[0130] 4. Stability testing of nano-astaxanthin liposomes in the cosmetic field may include the following steps:

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

[0132] Formula stability testing: Attention should be paid to whether there are chemical and / or biological interactions between the ingredients in the formula (serum, cream) and whether stratification occurs in the formula over time.

[0133] The essence water and face cream prepared according to Application Example 1 and Application Example 2 were placed under six conditions for 7 days: high temperature, freezing, alternating hot and cold, light exposure, room temperature, and refrigeration. The results were photographed and observed on the 1st, 3rd, 5th, and 7th days to see if the nano-astaxanthin liposomes or formula showed discoloration, fading, or layering over time.

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

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

[0136] C. Alternating hot and cold test: Store the sample in a sealed clean container, then place it in a refrigerator pre-adjusted to -20℃. After 24 hours, remove the sample and place it in a constant temperature incubator pre-adjusted to 50℃ for another 24 hours. Repeat this alternation process at 24-hour intervals for 7 days. Observe the sample after it returns to room temperature.

[0137] D-light irradiation test: Place an appropriate amount of sample in a suitable light-transmitting container under a transparent glass window, and take samples for observation on days 1, 3, 5, and 7. During the test, it is important to control the temperature to maintain a constant level with room temperature, and carefully observe any changes in the appearance of the samples.

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

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

[0140] The observation results show that the nano-astaxanthin liposomes showed almost no fading under all conditions. Figure 7 The essence water in the formula slightly fades under light exposure, and the 5% and 10% essence waters show slight fading under high temperature conditions. Figure 8 The face cream slightly faded under light exposure, and the 5% and 10% face creams showed slight fading under high temperature conditions. Figure 9 ).

[0141] The above results demonstrate that the nano-astaxanthin liposomes prepared by the present invention in a specific ratio 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. Furthermore, they can be applied in the cosmetic field, greatly improving the bioavailability of astaxanthin.

[0142] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several modifications or equivalent substitutions can be made to the technical solution without departing from the principle of the present invention, and these modifications or equivalent substitutions should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing nano-astaxanthin liposomes, characterized in that, Includes the following steps: (1) Haematococcus pluvialis extract, lecithin and cholesterol were dissolved in an organic solvent and reacted under heating conditions to obtain a lipid film containing astaxanthin; (2) Add hydration solvent and Tween-80 to the lipid film obtained in step (1), and sonicate to obtain the nano-astaxanthin liposomes; The mass ratio of the Haematococcus pluvialis extract, lecithin, and cholesterol is 40:250:

50. The total mass of Haematococcus pluvialis extract, lecithin and cholesterol in step (1) is in the volume ratio of hydrated solvent and Tween-80 in step (2) to 340 mg: 50 mL: 15 mL.

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

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

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

5. A nano-astaxanthin liposome prepared by the preparation method according to any one of claims 1-4.

6. The application of the nano-astaxanthin liposomes prepared by the preparation method according to any one of claims 1-4 in the preparation of cosmetics.

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