SOD (superoxide dismutase) liposome as well as preparation method and application thereof

Abstract

The invention discloses SOD (superoxide dismutase) lipidosome as well as a preparation method and application thereof, and belongs to the technical field of biotechnology or cosmetics. The preparation method of the SOD liposome comprises the following steps: fully dissolving lecithin and cholesterol with absolute ethyl alcohol, and then blow-drying the absolute ethyl alcohol to obtain a liposome precursor; and adding preheated absolute ethyl alcohol and an SOD solution into the liposome precursor, carrying out water bath heat preservation, and then cooling to room temperature. Compared with lipidosome obtained by a traditional film dispersion method, the SOD lipidosome obtained by the method has the advantages of high encapsulation efficiency, higher stability, higher transdermal absorption effect, higher bioavailability and more obvious anti-aging effect.

Description

Technical Field

[0001] The invention relates to a SOD liposome and a preparation method and application thereof, belonging to the field of biotechnology or cosmetic technology. Background Art

[0002] Superoxide dismutase, referred to as SOD, is the most important enzyme discovered so far to eliminate the superoxide ion free radical (O 2- It is an antioxidant enzyme that is the only scavenger of free radicals in the body and an inhibitor of tyrosinase, which plays an important role in the synthesis of melanin in the human body. After long-term basic theoretical research in the medical community, it is confirmed that the supplementation of SOD is of great benefit to the anti-aging of the human body. However, since SOD is a biological agent with a large molecular weight, its compatibility and application in preparations and its penetration in the skin are the most concerned and hottest topics in the cosmetics industry.

[0003] In transdermal delivery, liposomes, as a highly regarded nanoformulation, can not only enhance the compatibility of the package with the skin and promote transdermal delivery, but also improve the stability of the package and enhance compatibility with other matrix components and active ingredients. There have been some reports and applications of SOD liposomes in the early stage. The highest SOD liposome encapsulation rate reported in Rengel, EUR J PHARMACOL, 2002, 15: 441-448 is 35%. However, charged liposomes may cause certain damage during human absorption and cannot be used in food.

[0004] The preparation method of SOD liposomes reported at present is basically a thin film dispersion method, which is one of the most conventional technologies for preparing liposomes. Its preparation process is mainly divided into two steps: first drying the lipid dissolved in the organic solvent to form a lipid film, and dispersing the lipid film in an aqueous solution to self-assemble into a double-layer structure. Compared with the thin film dispersion method, other conventional preparation methods (such as reverse evaporation method, injection / evaporation method and dehydration-rehydration method, etc.) also have certain similarities, which are roughly divided into four steps: dissolving lipids in an organic solvent, evaporating and drying the organic solvent, dispersing lipids in an aqueous medium, and self-assembling to form liposomes. Usually, the liposomes prepared by the above-mentioned traditional methods will have one or more phospholipid bilayers. However, in the process of post-treatment, steps such as ultrasonic treatment are often used, which is not easy to amplify, and for macromolecular proteins such as SOD, the most reported method at present is the thin film dispersion method, such as Chinese patent CN113827491A, but this method has a low encapsulation rate and can only reach about 70% at most, and is unstable, so it is necessary to find a more economical and efficient method for preparing SOD liposomes. Summary of the invention

[0005] [Technical issues]

[0006] In the prior art, SOD liposomes are mostly prepared by thin film dispersion method. The SOD liposomes prepared by this traditional method have problems such as low encapsulation efficiency, poor stability, and poor bioavailability, which is not conducive to the deep processing and wide application of SOD.

[0007] [Technical solution]

[0008] In order to solve the defects of the prior art, the purpose of the present invention is to provide a SOD liposome and a preparation method and application thereof. The SOD liposome has a higher encapsulation rate than the liposome obtained by the traditional film dispersion method, is more stable, has a higher transdermal absorption effect and bioavailability, and has a more obvious anti-aging effect.

[0009] In order to achieve the above purpose, the technical solutions provided are as follows:

[0010] The present invention provides a method for preparing SOD liposomes, the method comprising the following steps:

[0011] (1) fully dissolving lecithin and cholesterol in anhydrous ethanol, and then drying the anhydrous ethanol to obtain a liposome precursor;

[0012] (2) Add the preheated anhydrous ethanol and SOD solution to the liposome precursor obtained in step (1), keep warm in a water bath, and then cool to room temperature.

[0013] In one embodiment, the lecithin in step (1) includes one or more of lecithin PC 80, lecithin PC 60, lecithin PC 20, hydrogenated lecithin, and egg yolk lecithin.

[0014] In one embodiment, the mass ratio of lecithin to cholesterol in step (1) is 700:100-500; preferably 700:300-500; further preferably 700:300-400.

[0015] In one embodiment, the dosage of the anhydrous ethanol in step (1) is sufficient to completely dissolve lecithin and cholesterol; the dosage of anhydrous ethanol is preferably 700:(100-500):5-15 ml; mg:mg:mL; more preferably: 700:(100-500):10; mg:mg:mL.

[0016] In one embodiment, the liposome precursor preparation process in step (1) also includes a surfactant.

[0017] In one embodiment, the surfactant includes one or more of Tween 20, Tween 60, Tween 80, PEG, glycerol, and palmitic acid; preferably Tween 20, Tween 60, and Tween 80; and more preferably Tween 80.

[0018] In one embodiment, the mass ratio of the lecithin, cholesterol and surfactant is 700:100 to 500:100.

[0019] In one embodiment, the preheated anhydrous ethanol in step (2) refers to anhydrous ethanol preheated to 50-60°C, preferably 60°C.

[0020] In one embodiment, the SOD solution in step (2) is any one of a SOD solution prepared in PBS, a SOD solution prepared in 1 / 10 PBS, and a SOD solution prepared in Tris-HCl; preferably, it is a SOD solution prepared in 1 / 10 PBS.

[0021] In one embodiment, the pH value of the PBS is 7.4.

[0022] In one embodiment, the final concentration of SOD in the SOD liposome is 3000 IU / ml to 30000 IU / ml; preferably 3000 IU / ml to 20000 IU / ml.

[0023] In one embodiment, the mass ratio of the liposome precursor, anhydrous ethanol and SOD solution in step (2) is 100:(50-100):(100-200); preferably 100:(50-80):(100-200); further preferably 100:80:200.

[0024] In one embodiment, the water bath insulation in step (2) is at a temperature of 40 to 60° C. and lasts for 5 to 20 minutes.

[0025] The present invention also provides the SOD liposome obtained by the above-mentioned preparation method.

[0026] The present invention also provides the use of the above-mentioned SOD liposome in skin care products such as emulsions, essences, and face creams.

[0027] The present invention also provides a freeze-dried powder of the SOD liposome described above, wherein the freeze-dried powder is obtained by mixing the SOD liposome and a freeze-drying protective agent and freeze-drying them together.

[0028] In one embodiment, the lyoprotectant is one or more of glucose, sucrose and PVP K60; preferably PVP K60 and sucrose; more preferably PVP K60.

[0029] In one embodiment, the mass fraction of the lyoprotectant is 5-10%, preferably 5%.

[0030] Beneficial effects:

[0031] The SOD liposomes prepared by the present invention have a higher encapsulation rate than the liposomes obtained by the traditional thin film dispersion method, and are the highest encapsulation rate reported so far:

[0032] (1) In the present invention, the encapsulation efficiency of SOD is increased to 92% by adding a surfactant, and the particle size is 170.2 nm, which is more stable;

[0033] (2) The transdermal absorption effect of the SOD liposomes in the present invention is 120% higher than that of the SOD aqueous solution;

[0034] (3) The bioavailability of SOD liposomes in the present invention is higher than that of SOD aqueous solution; the SOD group reaches a peak in feces at 4 hours, while the SOD-lipo group maintains a low level of continuous excretion, indicating that SOD-lipo is excreted less, the absorption capacity of the small intestine is stronger, and the bioavailability is higher;

[0035] (4) The anti-aging effect of SOD liposomes in the present invention is more significant than that of SOD aqueous solution; the epidermal thickness and dermal thickness of the SOD-lip group are significantly higher than those of the blank group, and the integrity of the skin and the number of hair follicles are significantly improved. Oral administration of SOD liposomes can significantly increase the collagen content in the skin of aging mice, making the collagen present a regular and dense arrangement. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The actual picture of the state of SOD liposome under the action of different lyophilization protective agents in Example 2;

[0037] Figure 2 The stability test results of SOD liposomes under different conditions in Example 3 are shown in FIG.

[0038] Figure 3 The transdermal absorption effect diagram of SOD liposomes under different conditions in Example 4;

[0039] Figure 4 The data diagram of SOD bioavailability under different conditions in Example 5;

[0040] Figure 5 These are H&E staining images of mouse skin under different treatment conditions in Example 5; (A) blank group; (B) SOD group; (C) SOD-lip group;

[0041] Figure 6 These are Masson's staining images of mouse skin under different treatment conditions in Example 5; (A) blank group; (B) SOD group; (C) SOD-lip group. DETAILED DESCRIPTION

[0042] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The following specific implementation methods further describe the present invention.

[0043] The culture medium used in the embodiment is:

[0044] 1×PBS: 10mmol / L Phosphate buffer; 137mmol / L NaCl; 2.7mmol / L KCl; pH 7.4.

[0045] Tris-HCl: 50 ml of 0.1 mol / L tris (hydroxymethyl) aminomethane (Tris) solution, adjust the pH to 7.4 with 0.1 mol / L hydrochloric acid, and dilute to 100 ml with water to obtain 0.05 mol / L Tris-HCl.

[0046] Solution A: pH 8.2 0.01mol / L, tris(hydroxymethyl)aminomethane (Tis)-hydrochloric acid buffer solution (containing 1mol / EDTA·2Na). Weigh 1.2114g Tis and 37.2mg EDTA·2Na and dissolve them in 62.4mL 0.1mol / L hydrochloric acid solution, and dilute to 100mL with distilled water.

[0047] Solution B: 4.5mmol / L pyrogallol hydrochloric acid solution; weigh 56.7mg of pyrogallol (AR) and dissolve it in a small amount of 10mmol / L hydrochloric acid solution, and make up to 100mL.

[0048] The test method involved in the present invention is:

[0049] 1. Encapsulation rate - 1 - (SOD enzyme activity in liposome solution / total SOD enzyme activity added) * 100%

[0050] The enzyme activity in liposomes was detected by the pyrogallol method. The specific method is as follows:

[0051] (1) Determination of the self-oxidation rate of pyrogallol

[0052] At about 25°C, add 2.35 mL of solution A, 2.00 mL of distilled water, and 0.15 mL of solution B into a 10 mL colorimetric tube at once. Mix solution B immediately and add it to the colorimetric dish. Measure the initial value and the absorbance value after 1 minute at a wavelength of 325 nm. The difference between the two is the self-oxidation rate of pyrogallol △A325 (min -1 ).

[0053] (2) Collect sample solution to measure the rate of inhibition of pyrogallol self-oxidation. According to the steps for measuring the rate of pyrogallol self-oxidation, add a certain amount of sample solution or enzyme solution to inhibit the rate of pyrogallol self-oxidation by about 1 / 2 △ A325 (min -1 ), that is, 325 (min -1 ) is 0.030.

[0054] The order of adding samples for SOD activity determination is shown in the table below:

[0055] Test solution Blank (auto-oxidation) Collect sample liquid Liquid A / mL 2.35 2.35 Distilled water / mL 2.00 1.98 Sample solution or SOD enzyme solution / mL -- 0.02 Liquid B / mL 0.15 0.15

[0056] Note: Liquid samples are calculated according to formula (1):

[0057]

[0058] Where:

[0059] U / mL——SOD enzyme activity unit;

[0060] ΔA 325 ——pyrogallol autoxidation rate;

[0061] ΔA′ 325 ——The sample solution or SOD alcohol solution inhibits the self-oxidation rate of pyrogallol;

[0062] V——volume of enzyme solution or sample solution added, in milliliters (mL);

[0063] D——Dilution multiple of enzyme solution or sample solution;

[0064] 4.5——Total volume of reaction solution, in milliliters (mL).

[0065] The calculation result shall be rounded to three significant figures.

[0066] Example 1 Preparation of SOD liposomes

[0067] The SOD liposomes were prepared by thin film dispersion method and liposome precursor method, respectively, which specifically included the following steps:

[0068] Thin film dispersion method: weigh 700 mg of lecithin PC 80 and 300 mg of cholesterol into a 1L eggplant-shaped bottle, add 100 mL of anhydrous ethanol to dissolve, put it in a 45°C water bath after dissolution, and use a rotary evaporator at 100 hPa to remove most of the anhydrous ethanol, vacuum dry for 2 hours to obtain a lipid honeycomb film; add 100.00 mL of 3000 IU / mL SOD aqueous solution at room temperature and rotate to hydrate until the lipid honeycomb film is completely dissolved, ultrasonicate at 4°C for 25 minutes, pass through a 0.45 μm filter membrane, measure the particle size and encapsulation efficiency, and store at 4°C.

[0069] Proliposome method: Weigh 700 mg of lecithin PC-80 and 300 mg of cholesterol, fully dissolve them with 10 ml of anhydrous ethanol, and then use a nitrogen blower at 65°C to blow off most of the ethanol to obtain a liposome prosome; add anhydrous ethanol preheated at 60°C and a SOD solution dissolved in PBS, and mix the liposome prosome, ethanol and SOD solution in a mass ratio of 100:80:200. After mixing, place in a 60°C water bath for 10 minutes and slowly cool to room temperature; then add PBS dropwise to 100 mL at room temperature and 600 r / min to make the final SOD concentration of 3000 IU / ml, filter with a 0.45 μm filter membrane, measure the particle size and encapsulation efficiency, and store at 4°C.

[0070] The encapsulation efficiency results of different liposomes are shown in Table 1:

[0071] Table 1: Test results of different liposome preparation methods

[0072]

[0073] The results are shown in Table 1. The encapsulation efficiency of liposomes prepared by the proliposome method is 82%, while the encapsulation efficiency of liposomes prepared by the thin film dispersion method is only 15%. At the same time, the particle size of liposomes prepared by the proliposome method is smaller and more uniform.

[0074] Example 2 SOD liposome optimization

[0075] 1. Optimization of different contents of lecithin and cholesterol

[0076] Weigh lecithin and cholesterol according to Table 2, fully dissolve them with 10 ml of anhydrous ethanol, then use a nitrogen blower to blow off most of the ethanol at 65°C to obtain a liposome precursor, add anhydrous ethanol preheated at 60°C and a SOD solution dissolved in PBS, and mix the liposome precursor, ethanol and SOD solution in a mass ratio of 100:80:200; after mixing evenly, place in a 60°C water bath for 10 minutes and slowly cool to room temperature; then add PBS dropwise to 100 mL at room temperature and 600 r / min to make the final SOD concentration 3000 IU / ml, filter with a 0.45 μm filter membrane, and measure the encapsulation efficiency and particle size.

[0077] Table 2 Detection results of different proportions of lecithin and cholesterol in 100ml liposome system

[0078]

[0079] The test results show that the liposomes obtained according to the ratio of Experiment 2 and Experiment 3 have the highest encapsulation rate and relatively small particle size, among which Experiment 3 has the highest encapsulation rate, that is, the optimal ratio of lecithin and cholesterol in a 100ml system is 7:3, in which the amount of lecithin is 700mg and the amount of cholesterol is 300mg.

[0080] 2. Surfactant Optimization

[0081] Weigh 700mg lecithin, 300mg cholesterol and 100mg surfactant, fully dissolve them with 10ml anhydrous ethanol, then use a nitrogen blower at 65℃ to blow most of the ethanol to obtain liposome precursors, add anhydrous ethanol preheated at 60℃ and SOD solution dissolved in PBS, and mix the liposome precursors, ethanol and SOD solution in a mass ratio of 100:80:200. After mixing evenly, place in a 60℃ water bath for 10min and slowly cool to room temperature. Then, add PBS to 100mL at room temperature and 600r / min to make the final SOD concentration 3000IU / ml, and measure the particle size and encapsulation efficiency.

[0082] Table 3 Test results of different surfactants

[0083]

[0084]

[0085] The test results showed that the liposomes with the surfactant added in Experiment 3 had the highest encapsulation efficiency and a relatively small particle size.

[0086] 3. Ratio Optimization

[0087] Weigh 700 mg of lecithin, 300 mg of cholesterol and 100 mg of Tween 80, fully dissolve them with 10 ml of anhydrous ethanol, then use a nitrogen blower to blow most of the ethanol to dry at 65 ° C to obtain a liposome precursor, add anhydrous ethanol preheated at 60 ° C and SOD solution dissolved in PBS, and mix the liposome precursor, ethanol and SOD solution in a mass ratio of 100:80:100, 100:80:200, 100:50:100, 100:50:200, 100:100:100, 100:100:200; after mixing evenly, place in a 60 ° C water bath for 10 minutes and slowly cool to room temperature. Then, add PBS to 100 mL at room temperature and 600 r / min to make the final SOD concentration 3000 IU / ml, and measure the particle size and encapsulation efficiency.

[0088] Table 4 Detection results of different proportions

[0089] Proportion Encapsulation efficiency Particle size (nm) 1 100:80:100 78.00％ 180.5 2 100:80:200 92.00％ 170.2 3 100:50:100 70.30％ 190.8 4 100:50:200 65.60％ 264.4 5 100:100:100 72.00％ 200.5 6 100:100:200 73.60％ 210.3

[0090] The test results showed that the liposomes obtained according to the ratio of Experiment 2 had the highest encapsulation rate and a relatively small particle size.

[0091] 4. SOD concentration optimization

[0092] Weigh 700 mg of lecithin, 300 mg of cholesterol and 100 mg of Tween 80, fully dissolve them with 10 ml of anhydrous ethanol, then use a nitrogen blower to dry most of the ethanol at 65 ° C to obtain a liposome precursor, add anhydrous ethanol preheated at 60 ° C and a SOD solution dissolved in PBS, and mix the liposome precursor, ethanol and SOD solution in a mass ratio of 100:80:200; after mixing evenly, place in a 60 ° C water bath for 10 min and slowly cool to room temperature; then add PBS dropwise to 100 mL at room temperature and 600 r / min to make the final SOD concentrations of 3000, 5000, 10000, 20000, 30000, and 50000 IU / ml, respectively, and measure the particle size and encapsulation efficiency.

[0093] Table 5 Detection results of different SOD concentrations

[0094]

[0095]

[0096] The results showed that the liposomes could encapsulate up to 20,000 IU / ml of SOD, with an encapsulation rate of 92.2%. When the SOD enzyme activity increased to 30,000 IU / ml, the encapsulation rate dropped to 82.60%.

[0097] 5. Water Phase Optimization

[0098] Weigh 700 mg of lecithin, 300 mg of cholesterol and 100 mg of Tween 80, fully dissolve them with 10 ml of anhydrous ethanol, then use a nitrogen blower to blow off most of the ethanol at 65°C to obtain a liposome precursor, add anhydrous ethanol preheated at 60°C and a SOD solution dissolved in PBS, 1 / 10 PBS, and pH 7.4 Tris-HCl, and mix the liposome precursor, ethanol and SOD solution in a mass ratio of 100:80:200; after mixing evenly, place in a 60°C water bath for 10 minutes and slowly cool to room temperature; then, add PBS, 1 / 10 PBS, and Tris-HCl dropwise to 100 mL at room temperature and 600 r / min to make the final SOD concentration 20000 IU / ml, and measure the particle size and encapsulation efficiency.

[0099] Table 6 Detection results of different water phases

[0100] Water Phase Encapsulation efficiency Particle size (nm) 1 PBS 92.00％ 170.2 2 1 / 10 PBS 95.50％ 166.5 3 Tris-HCl 87.80％ 190.5

[0101] The results showed that the liposomes obtained by using 1 / 10 PBS as the aqueous phase had an encapsulation efficiency of up to 95.5%.

[0102] 6. Optimization of freeze-drying protective agent

[0103] Weigh 700mg lecithin, 300mg cholesterol and 100mg Tween 80, fully dissolve with 10ml anhydrous ethanol, then use a nitrogen blower to blow most of the ethanol to dry at 65℃ to obtain liposome precursor, add anhydrous ethanol preheated at 60℃ and 1 / 10PBS, dissolved SOD solution, and mix the liposome precursor, ethanol and SOD solution in a mass ratio of 100:80:200. After mixing evenly, place in a 60℃ water bath for 10min and slowly cool to room temperature; then add 1 / 10PBS to 100mL at room temperature and 600r / min to make the final SOD concentration 20000IU / ml, and add 5% glucose, 10% sucrose and 5% PVPK60 to the liposomes for freeze-drying.

[0104] The freeze-dried state Figure 1 As shown in the figure, the liposomes with 5% PVP K60 are in the best condition, followed by those with 10% sucrose, with some of them sticking to the bottom, and most of them sticking to the bottom with 5% glucose. The encapsulation rate remains unchanged after re-dissolution.

[0105] Example 3 SOD liposome stability

[0106] The experiment was divided into three groups: 1. Liposomes prepared by thin film dispersion method; 2. Liposomes prepared by proliposome method; 3. SOD aqueous solution; these three groups of SOD were placed at 4℃, 25℃, 50℃ and UV conditions to detect stability, detection index: SOD enzyme activity; detection time points: D0, D7, D14, D21, D30, D60, D90.

[0107] The results are as follows Figure 2 As shown: the liposomes prepared by the proliposome method under the four conditions can remain stable, while the activity of the liposomes prepared by the SOD aqueous solution and the film dispersion method gradually decreases with the extension of time.

[0108] Example 4 SOD liposome transdermal absorption

[0109] The experiment was divided into 4 groups: 1. Liposomes prepared by thin film dispersion method; 2. Liposomes prepared by proliposome method; 3. SOD aqueous solution; 4. Blank. These 4 groups were added into the commercial brand basic emulsion at 1wt% to make the final concentration 2000IU / ml.

[0110] a. Install the diffusion cell

[0111] Place the diffusion cell in the dry heating cell, and insert the connecting pipes and the signal port of the temperature control probe into the rear interface of the dry heating cell. Cut the pigskin of appropriate size and place it flatly into the inner circle of the silicone gasket, cover the diffusion cell cover in the center, put on the anti-evaporation glass sheet, fix the diffusion cell and tighten the fixing nut.

[0112] b. Add receiving solution

[0113] Use a syringe to slowly add the receiving solution PBS into the receiving cell. After adding, check whether there are bubbles in the receiving cell. If there are bubbles, slightly tilt the diffusion cell to expel the bubbles and fill it with the receiving solution.

[0114] c. Add product solution and set the program

[0115] Open the anti-evaporation glass sheet and slowly add the product solution into the supply tank with a syringe. The amount added is 1 mL. After adding, cover the anti-evaporation glass sheet. Manually control the head to lift and lower bubbles and observe the leakage, set the temperature, turn on heating and stirring, and then start the operation.

[0116] d. Sampling

[0117] Collect the sample solution 0.1h, 0.5h, 1h, 2h, 4h, 6h, 8h, 12h, and 24h after adding the sample. Insert a 5mL syringe into the sampling port and draw 3mL of solution into the centrifuge tube. Use a syringe to add the receiving solution PBS. After adding, check whether there are bubbles in the receiving cell. If there are bubbles, tilt the diffusion cell slightly to expel the bubbles and fill it with the receiving solution.

[0118] e. SOD activity assay

[0119] The results are as follows Figure 3As shown: 24 hours after the liposomes penetrated the skin, the highest transdermal absorption rate of the liposomes prepared by the proliposome method in experiment 2 reached 38.5%, followed by the liposomes prepared by the thin film dispersion method in experiment 1, which reached a highest transdermal absorption rate of 20.5% after 24 hours, followed by experiment 3, which reached a highest transdermal absorption rate of 17.5% after 24 hours.

[0120] Example 5 SOD liposome bioavailability

[0121] a. Mouse strain: C57BL / 6 mice (Source: Zhuhai Baishitong Biotechnology Co., Ltd., age: 6 weeks, weight: 20-22g)

[0122] b. Grouping (n=10): 1. Blank group: normal saline; 2. SOD group: SOD aqueous solution 10000 IU / mouse; 3. SOD-lip (proliposome method) group: SOD liposome 10000 IU / mouse;

[0123] c. Gavage, collecting feces at 0, 1, 2, 4, 6, 8, 10, 12, and 24 hours, and detecting SOD enzyme activity in feces by pyrogallol method;

[0124] e. Pathological sections: skin H&E staining, Masson's staining;

[0125] After mice were orally administered with SOD aqueous solution and SOD-lipo, the SOD content in feces was detected. Figure 4 The results showed that the SOD group reached a peak in feces after 4 hours, while the SOD-lipo group maintained a low level of continuous excretion, indicating that SOD-lipo was excreted less, the small intestine had a stronger absorption capacity, and had a higher bioavailability.

[0126] H&E staining can show the state of skin tissue. Figure 5 As shown, the epidermal thickness and dermal thickness of the SOD group and SOD-lip group were significantly higher than those of the blank group, and the skin integrity and the number of hair follicles were significantly improved, and the number of hair follicles in the SOD-lip group was significantly greater than that in the SOD group. This indicates that SOD liposomes have a better anti-skin aging effect.

[0127] Masson's staining can reflect the content of collagen in the skin. The higher the collagen content, the darker the blue-purple color. Figure 6As shown, the collagen content of the skin of mice in the blank group was the lowest, and it was in an irregular and loose state; the collagen content of the skin of mice in the SOD group increased, and the collagen content of the skin of mice in the SOD-lip group was the highest, with regular fiber arrangement and a dense state. The comprehensive results show that oral SOD liposomes can significantly increase the collagen content of the skin of aging mice, making the collagen present a regular and dense arrangement.

[0128] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing SOD liposomes, characterized in that: The method comprises the following steps: (1) fully dissolving lecithin and cholesterol in anhydrous ethanol, and then drying the anhydrous ethanol to obtain a liposome precursor; (2) Add the preheated anhydrous ethanol and SOD solution to the liposome precursor obtained in step (1), keep warm in a water bath, and then cool to room temperature.

2. The preparation method according to claim 1, characterized in that: The mass ratio of lecithin to cholesterol in step (1) is 700:100-500.

3. The preparation method according to claim 1, characterized in that: The liposome precursor preparation process in step (1) also includes a surfactant.

4. The preparation method according to claim 3, characterized in that: The surfactant includes one or more of Tween 20, Tween 60, Tween 80, PEG, glycerol, and palmitic acid.

5. The preparation method according to claim 1, characterized in that: The SOD solution in step (2) is any one of a SOD solution prepared in PBS, a SOD solution prepared in 1 / 10 PBS, and a SOD solution prepared in Tris-HCl.

6. The preparation method according to claim 1, characterized in that: The final concentration of SOD in the SOD liposome is 3000 IU / ml to 30000 IU / ml.

7. The preparation method according to claim 1, characterized in that: The mass ratio of the liposome precursor, anhydrous ethanol and SOD solution in step (2) is 100:(50-100):(100-200).

8. The SOD liposome obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the SOD liposome according to claim 8 in preparing emulsion, essence, cream and skin care products.

10. A freeze-dried powder of SOD liposomes, characterized in that: The lyophilized powder is prepared by mixing the SOD liposomes described in claim 8 with a lyophilization protectant and lyophilizing them together.

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