A ferulic acid lyosome with multiple anti-aging effects, its preparation method, applications, and products.

Abstract

This invention relates to the field of cosmetic technology, specifically to a ferulic acid liposome with multiple anti-aging effects, its preparation method, application, and product. The ferulic acid liposome comprises, by weight, 80-100 parts soy lecithin, 5 parts astragaloside A, 5 parts ceramide, and 10-15 parts ferulic acid. The preparation method of the ferulic acid liposome includes the following steps: A) mixing the soy lecithin, astragaloside A, ceramide, and ferulic acid in the prescribed amounts to obtain a mixture; adding anhydrous ethanol to the mixture and stirring until dissolved to obtain an organic phase; B) injecting the organic phase obtained in step A into water for hydration to obtain a solution; C) filtering the solution obtained in step B to obtain the ferulic acid liposome. The ferulic acid liposome of this invention avoids the risk of cardiovascular and cerebrovascular diseases while improving carrier stability and providing more skincare benefits.

Description

Technical Field

[0001] This invention relates to the fields of cosmetics and skin care, specifically to a ferulic acid lyosome with multiple anti-aging effects, its preparation method, applications, and products. Background Technology

[0002] The skin is the outermost protective organ of the human body, serving as the body's first natural barrier against external aggressors and playing a crucial role in defending against environmental attacks. The skin consists of two layers: the epidermis and the dermis. The epidermis, located on the skin's surface, can be further divided into the stratum corneum and the stratum germinativum. The stratum corneum is composed of mature keratinocytes and intercellular lipids. The keratinocytes act as the "bricks," while the intercellular lipids act as the "mortar," forming a stable, brick-wall-like structure that ensures the integrity of the skin barrier. The intercellular lipids in the stratum corneum, composed of approximately 50% ceramides, 25% cholesterol, and 15% free fatty acids, are essential components that help keratinocytes adhere tightly and maintain the skin barrier function. The health of the skin barrier is closely related to these intercellular lipids; any abnormalities or deficiencies can damage the skin barrier function and lead to skin problems.

[0003] Ferulic acid (FA) is a phenolic acid compound widely found in the plant kingdom. It is a major component of plant cell walls, cross-linked with cell wall polysaccharides and lignin. Ferulic acid is an active ingredient in many foods, such as wheat bran, rice bran, and other cereals; citrus fruits, bananas, and other fruits; and eggplants and broccoli, all rich sources of ferulic acid. Ferulic acid has multiple anti-aging effects on the human body, including antioxidant, whitening, anti-inflammatory, anti-cancer, sun protection, moisturizing, and repairing properties. Therefore, ferulic acid has a very broad application prospect in the cosmetics industry. However, due to its poor solubility and stability, its absorption and bioavailability by human skin are low. Furthermore, light, heat, oxidation, and acidic environments can easily lead to a decrease in the efficacy of ferulic acid under various conditions, thus limiting its application. Emulsification systems such as nanoemulsions, liposomes, and self-assembled micelles can be used to solve these problems, as illustrated by Chinese patents CN115634167B and CN117159431A.

[0004] Because traditional emulsification techniques often only reach the stratum corneum of the skin, making it difficult to deliver active ingredients to the deeper layers, Touitou et al. developed a novel carrier composed of phospholipids, ethanol, and water, which they defined as ethosomal bodies (ES). Ethosomes are liposomes with the addition of 20%-50% (w / w) ethanol, and also possess the ability to encapsulate both hydrophilic and hydrophobic compounds within them.

[0005] For example, Deng Mengjie et al. published an article entitled "Preparation, Characterization and In Vitro Transdermal Absorption Study of Ferulic Acid Nanoparticles" in the journal "Daily Chemical Industry (Chinese and English)" in 2023. This article studied the transdermal delivery and antioxidant properties of ferulic acid nanoparticles prepared by microfluidic technology. First, the effect of lecithin and edge activators (sucrose palmitate and Tween-80) on the encapsulation efficiency of ferulic acid nanoparticles was studied using response surface methodology. The optimization results showed that when the ratio of lecithin, sucrose palmitate and Tween-80 was 0.68%:0.51%:0.25% (w / %), the prepared ferulic acid nanoparticles had small and uniform particle size (104.5±0.7 nm), high encapsulation efficiency (88.8%±2.6%), and high stability (stable for 60 days at 25℃). Secondly, the antioxidant and transdermal delivery properties of ferulic acid nanoparticles were evaluated using DPPH free radical scavenging assays and Franz diffusion cell in vitro transdermal assays, respectively. The study found that the prepared ferulic acid nanoparticles exhibited uniform particle size, high encapsulation efficiency, good stability, high antioxidant capacity, and good transdermal absorption performance, showing promise for use in transdermal delivery systems of phenolic acid natural active ingredients and cosmetics with antioxidant, anti-aging, and whitening effects. Although the ferulic acid nanoparticles prepared in this article showed excellent encapsulation efficiency and stability, the preparation process was complex. However, a major drawback of nanoparticles is the relatively high fluidity of the phospholipid bilayer, which was not specifically addressed in this study; therefore, it is necessary to find a component that can reduce the fluidity of the nanoparticles.

[0006] Secondly, cholesterol, as a stabilizer of the fluidity of the phospholipid bilayer molecular membrane, is used to increase the rigidity of the phospholipid bilayer. However, excessive absorption of cholesterol in the blood can lead to cardiovascular and cerebrovascular diseases such as hyperlipidemia. Therefore, it is urgent to find a component that can replace cholesterol as a stabilizer and also has anti-aging effects.

[0007] In summary, there is an urgent need for a ferulic acid lyosome with simple preparation method, low fluidity, good stability, high transdermal absorption rate, whitening, antioxidant, skin repair and multiple anti-aging effects, as well as its preparation method, application and products. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention aims to provide a ferulic acid liposome with a simple preparation method, low flowability, good stability, high transdermal absorption rate, whitening effect, and multiple anti-aging benefits, as well as its preparation method, applications, and products. The ferulic acid liposome comprises: soybean lecithin, astragaloside A, ceramide, and ferulic acid. Experiments have demonstrated that this ferulic acid liposome not only exhibits good stability and uniform particle size, but also high transdermal absorption rate, significantly inhibits melanin synthesis, and demonstrates whitening and antioxidant effects.

[0009] To achieve the above objectives, the present invention employs the following technical solution:

[0010] On the one hand, the present invention provides a ferulic acid liposome with multiple anti-aging effects, wherein the ferulic acid liposome comprises, by weight parts: 80-100 parts soybean lecithin, 5 parts astragaloside A, 5 parts ceramide and 10-15 parts ferulic acid.

[0011] More preferably, the mass ratio of soybean lecithin, astragaloside A, ceramide, and ferulic acid is 18:1:1:2.

[0012] Secondly, the present invention also provides a method for preparing the above-mentioned ferulic acid liposomes with multiple anti-aging effects, characterized by comprising the following steps:

[0013] A. Mix the soybean lecithin, astragaloside A, ceramide and ferulic acid according to the formula to obtain a mixture. Add solvent to the mixture and stir until dissolved to obtain an organic phase.

[0014] B. Inject the organic phase obtained in step A into the aqueous phase and hydrate it to obtain a solution;

[0015] C. Filter the solution obtained in step B to obtain the ferulic acid alcohol body.

[0016] Preferably, the solvent in step A is anhydrous ethanol; the stirring method is constant temperature stirring, and the constant temperature is 40-50℃; more preferably, the temperature is 45℃.

[0017] Preferably, the mass-to-volume ratio of anhydrous ethanol to the mixture in step A is 100-120 mg: 1.5 mL.

[0018] More preferably, the mass-to-volume ratio of anhydrous ethanol to the mixture in step A is 110 mg: 1.5 mL.

[0019] Preferably, the aqueous phase in step B is pure water; the instrument used for hydration is a constant temperature magnetic stirrer, the hydration speed is 800 rpm, the hydration temperature is 45°C, and the hydration time is 20 min.

[0020] Preferably, the volume ratio of the aqueous phase to the organic phase in step B is 2-3:1.

[0021] More preferably, the volume ratio of the aqueous phase to the organic phase in step B is 2.3:1.

[0022] Preferably, the tool used when injecting the organic phase into the aqueous phase in step B is an injection needle.

[0023] Preferably, the filter used in step C is an organic microporous membrane with a pore size of 0.22 μm, and the filtration is performed three times.

[0024] Thirdly, the present invention also provides the application of the above-mentioned ferulic acid glycosides with multiple anti-aging effects in the preparation of products with multiple anti-aging effects.

[0025] Fourthly, the present invention also provides a product containing ferulic acid liposomes with multiple anti-aging effects. Preferably, the product is in the form of an emulsion.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] (1) In this invention, ferulic acid has better solubility and stability, and can deliver active ingredients to the deep layers of the skin. It is flexible and has high transdermal absorption efficiency.

[0028] (2) One of the main drawbacks of liposomes is that the phospholipid bilayer has relatively high fluidity. The present invention adds ceramide as a membrane modifier, which can reduce the fluidity of liposomes, improve their stability and enhance their transdermal permeability to the skin.

[0029] (3) This invention uses astragaloside A to replace cholesterol, which not only avoids the occurrence of cardiovascular and cerebrovascular diseases, but also brings more skin care benefits to liposomes.

[0030] (4) In this invention, astragaloside A and ceramide are added to liposomes as membrane modifiers to encapsulate ferulic acid, thereby achieving multiple anti-aging effects. Attached Figure Description

[0031] Figure 1 Images of FA-ES placed at different temperatures for 30 days;

[0032] Figure 2 Particle size and dispersion index (PDI) of FA-ES stored at different temperatures for 30 days;

[0033] Figure 3 Zeta potentials after 30 days at different temperatures;

[0034] Figure 4 DPPH free radical scavenging capacity diagram;

[0035] Figure 5 A fluorescence microscope image of pyrene in nematodes;

[0036] Figure 6 A diagram showing the skin transdermal permeability of nematodes;

[0037] Figure 7 A diagram showing the lifespan of nematodes;

[0038] Figure 8 Microscopic image of lipofuscin in nematodes;

[0039] Figure 9A graph showing the lipofuscin levels in nematodes;

[0040] Figure 10 A diagram showing the oxidative stress in nematodes;

[0041] Figure 11 A graph showing the total number of eggs laid by nematodes over 7 days;

[0042] Figure 12 A diagram illustrating the high-temperature stress response of nematodes;

[0043] Figure 13 A diagram showing the irritation of zebrafish embryos;

[0044] Figure 14 and 15 A graph showing the moisture retention rate of zebrafish;

[0045] Figure 16 A diagram showing oxidative stress in zebrafish;

[0046] Figure 17 and 18 A restored image of a zebrafish;

[0047] Figure 19 and 20 A diagram showing the staining degree of β-galactosidase in zebrafish;

[0048] Figure 21 and 22 This is a graph showing the melanin content of zebrafish. Detailed Implementation

[0049] The present invention will be further described in detail below with reference to specific embodiments.

[0050] Example 1

[0051] The ferulic acid alcohol body, by mass fraction, specifically comprises: 90 parts soybean lecithin, 5 parts astragaloside A, 5 parts ceramide, and 10 parts ferulic acid.

[0052] The preparation method of ferulic acid lyosomes is as follows:

[0053] A: The ethanol injection method was used to prepare the ethanol phase. First, 90 mg of soybean lecithin, 5 mg of astragaloside A, 5 mg of ceramide and 10 mg of ferulic acid were weighed in a vial and dispersed in 1.5 mL of anhydrous ethanol. The mixture was stirred and dissolved at a constant temperature of 45 °C to obtain the organic phase.

[0054] B: Use pure water as the aqueous phase. After the organic phase is completely dissolved, slowly inject 1.5 mL of the organic phase into 3.5 mL of the aqueous phase using a 2 mL syringe. Hydrate at 800 rpm and 45 °C for 20 min.

[0055] C: Finally, filter through a 0.22μm microporous membrane three times to obtain FA-ES.

[0056] Example 2

[0057] The ferulic acid alcohol body, by mass fraction, specifically comprises: 80 parts soybean lecithin, 5 parts astragaloside A, 5 parts ceramide, and 10 parts ferulic acid.

[0058] The preparation method of ferulic acid lyosomes is as follows:

[0059] A: The ethanol injection method was used to prepare the liposome. First, 80 mg of soybean lecithin, 5 mg of astragaloside A, 5 mg of ceramide and 10 mg of ferulic acid were weighed in a vial and dispersed in 1.5 mL of anhydrous ethanol. The mixture was stirred and dissolved at a constant temperature of 45 °C to obtain the organic phase.

[0060] B: Use pure water as the aqueous phase. After the organic phase is completely dissolved, slowly inject 1.5 mL of the organic phase into 3.5 mL of the aqueous phase using a 2 mL syringe. Hydrate at 800 rpm and 45 °C for 20 min.

[0061] C: Finally, filter through a 0.22μm microporous membrane three times to obtain FA-ES.

[0062] Example 3

[0063] The ferulic acid alcohol body specifically comprises, by weight, 100 parts soybean lecithin, 5 parts astragaloside A, 5 parts ceramide, and 10 parts ferulic acid.

[0064] The preparation method of ferulic acid lyosomes is as follows:

[0065] A: The ethanol injection method was used to prepare the liposome. First, 100 mg of soybean lecithin, 5 mg of astragaloside A, 5 mg of ceramide and 10 mg of ferulic acid were weighed in a vial and dispersed in 1.5 mL of anhydrous ethanol. The mixture was stirred and dissolved at a constant temperature of 45 °C to obtain the organic phase.

[0066] B: Use pure water as the aqueous phase. After the organic phase is completely dissolved, slowly inject 1.5 mL of the organic phase into 3.5 mL of the aqueous phase using a 2 mL syringe. Hydrate at 800 rpm and 45 °C for 20 min.

[0067] C: Finally, filter through a 0.22μm microporous membrane three times to obtain FA-ES.

[0068] Example 4

[0069] The ferulic acid alcohol body, by mass fraction, specifically comprises: 90 parts soybean lecithin, 5 parts astragaloside A, 5 parts ceramide, and 15 parts ferulic acid.

[0070] The preparation method of ferulic acid lyosomes is as follows:

[0071] A: The ethanol injection method was used to prepare the ethanol phase. First, 90 mg of soybean lecithin, 5 mg of astragaloside A, 5 mg of ceramide and 15 mg of ferulic acid were weighed in a vial and dispersed in 1.5 mL of anhydrous ethanol. The mixture was stirred and dissolved at a constant temperature of 45 °C to obtain the organic phase.

[0072] B: Use pure water as the aqueous phase. After the organic phase is completely dissolved, slowly inject 1.5 mL of the organic phase into 3.5 mL of the aqueous phase using a 2 mL syringe. Hydrate at 800 rpm and 45 °C for 20 min.

[0073] C: Finally, filter through a 0.22μm microporous membrane three times to obtain FA-ES.

[0074] The preparation method is the same as in Example 1.

[0075] Example 5

[0076] The composition and mass fraction of ferulic acid alcohol body are the same as in Example 1.

[0077] The preparation method of ferulic acid lyosomes is as follows:

[0078] A: The ethanol injection method was used to prepare the ethanol phase. First, 90 mg of soybean lecithin, 5 mg of astragaloside A, 5 mg of ceramide and 15 mg of ferulic acid were weighed in a vial and dispersed in 1.25 mL of anhydrous ethanol. The mixture was stirred and dissolved at a constant temperature of 45 °C to obtain the organic phase.

[0079] B: Use pure water as the aqueous phase. After the organic phase is completely dissolved, slowly inject 1.25 mL of the organic phase into 3.75 mL of the aqueous phase using a 2 mL syringe. Hydrate at 800 rpm and 45 °C for 20 min.

[0080] C: Finally, filter through a 0.22μm microporous membrane three times to obtain FA-ES.

[0081] Example 6

[0082] The composition and mass fraction of ferulic acid alcohol body are the same as in Example 1.

[0083] The preparation method of ferulic acid lyosomes is as follows:

[0084] A: The ethanol injection method was used to prepare the liposome. First, 90 mg of soybean lecithin, 5 mg of astragaloside A, 5 mg of ceramide and 15 mg of ferulic acid were weighed in a vial and dispersed in 1.75 mL of anhydrous ethanol. The mixture was stirred and dissolved at a constant temperature of 45 °C to obtain the organic phase.

[0085] B: Use pure water as the aqueous phase. After the organic phase is completely dissolved, slowly inject 1.75 mL of the organic phase into 3.25 mL of the aqueous phase using a 2 mL syringe. Hydrate for 20 min at 800 rpm and 45 °C.

[0086] C: Finally, filter through a 0.22μm microporous membrane three times to obtain FA-ES.

[0087] Comparative Example 1

[0088] The ferulic acid alcohol body, by mass fraction, specifically comprises: 90 parts soybean lecithin, 6.25 parts astragaloside A, 3.75 parts ceramide, and 10 parts ferulic acid.

[0089] The preparation method of ferulic acid lyosomes is as follows:

[0090] A: The ethanol injection method was used to prepare the liposome. First, 90 mg of soybean lecithin, 6.25 mg of astragaloside A, 3.75 mg of ceramide and 10 mg of ferulic acid were weighed in a vial and dispersed in 1.5 mL of anhydrous ethanol. The mixture was stirred and dissolved at a constant temperature of 45 °C to obtain the organic phase.

[0091] The remaining steps are the same as in Example 1.

[0092] Comparative Example 2

[0093] The ferulic acid alcohol body, by mass fraction, specifically comprises: 90 parts soybean lecithin, 3.75 parts astragaloside A, 6.25 parts ceramide, and 10 parts ferulic acid.

[0094] The preparation method of ferulic acid lyosomes is as follows:

[0095] A: The ethanol injection method was used to prepare the ethanol phase. First, 90 mg of soybean lecithin, 3.75 mg of astragaloside A, 6.25 mg of ceramide and 10 mg of ferulic acid were weighed in a vial and dispersed in 1.5 mL of anhydrous ethanol. The mixture was stirred and dissolved at a constant temperature of 45 °C to obtain the organic phase.

[0096] The other steps are the same as in Example 1.

[0097] Comparative Example 3

[0098] The ferulic acid alcohol body, by mass fraction, specifically comprises: 90 parts soybean lecithin, 5 parts astragaloside A, 5 parts ceramide, and 20 parts ferulic acid.

[0099] The preparation method of ferulic acid lyosomes is as follows:

[0100] A: The ethanol injection method was used to prepare the liposome. First, 90 mg of soybean lecithin, 5 mg of astragaloside A, 5 mg of ceramide and 20 mg of ferulic acid were weighed in a vial and dispersed in 1.5 mL of anhydrous ethanol. The mixture was stirred and dissolved at a constant temperature of 45 °C to obtain the organic phase.

[0101] The other steps are the same as in Example 1.

[0102] Effect Experiment

[0103] 1. Characterization of ferulic acid glycosomes

[0104] 1.1 Experimental Methods

[0105] Encapsulation efficiency determination: Step a: Separate FA-ES and FA using ultrafiltration centrifugation. Take 0.5 mL of freshly prepared FA-ES and place it in a 10 mL volumetric flask. Add an appropriate amount of anhydrous ethanol, sonicate to break the emulsion for 15 min, and then add anhydrous ethanol to make up to the volume. After filtering through a 0.22 μm microporous membrane, determine the total FA content (W) at a wavelength of 320 nm using a UV-Vis spectrophotometer. t Step b: Accurately transfer 0.5 mL of the same FA-ES into an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa. Centrifuge at 12,000 rpm for 15 min at 4 °C. Collect the filtrate and measure the free FA content (W) at the same wavelength. f ), calculate the encapsulation ratio using the following formula:

[0106]

[0107] Determination of particle size, PDI, and Zeta potential: Step a: Take the freshly prepared FA-ES solution and dilute it with pure water. Step b: Use a Malvern particle size potentiometer to measure the particle size, PDI, and Zeta potential of FA-ES at a scattering angle of 90° and a temperature of 25°C. Each sample is measured in triplicate.

[0108] Determination of storage stability: Step a: Take freshly prepared FA-ES solution, place it in a vial, and store it at 4℃, 25℃ and 37℃ for 30 days. Step b: Use a Malvern particle size potentiometer to measure the particle size, PDI and Zeta potential of FA-ES at a scattering angle of 90° and a temperature of 25℃ to investigate the storage stability of FA-ES.

[0109] 1.2 Experimental Results

[0110] like Figure 1 , 2 As shown in Tables 1 and 3, the effects of various factors on ethanol esters were investigated using particle size, PDI, Zeta potential, and encapsulation efficiency as indicators, and the process was optimized. Figure 1 , Figure 2 , Figure 3 As shown, after being placed at 4℃, 25℃, and 37℃ for 30 days, the FA-ES of Example 1 showed good stability in particle size, PDI, and Zeta potential compared to the comparative example. However, the encapsulation efficiency of the comparative example was not as good as that of the example, indicating that a better encapsulation efficiency is achieved when the mass ratio of ceramide to astragaloside A is 1:1 and the components contained in the ethanol body are within the limits specified in this invention. The optimal preparation conditions were finally determined as follows: soybean lecithin 90 mg, astragaloside A 5 mg, ceramide 5 mg, ferulic acid 10 mg, anhydrous ethanol volume fraction 30%, hydration temperature 45℃, hydration speed 800 rpm, hydration time 20 min, resulting in an ethanol body encapsulation efficiency of 52.87±1.56%, a particle size of 147.8±0.72 nm, a PDI of 0.12±0.01, and a Zeta potential of -32.3±0.90 mV.

[0111] Table 1

[0112]

[0113]

[0114] 2. In vitro antioxidant experiment

[0115] Based on Experiment 1, the FA-ES obtained by the components and preparation method of Example 1 was selected for subsequent experiments.

[0116] 2.1 DPPH Free Radical Scavenging Experimental Method

[0117] Step a: Mix 2 mL of 50 μg / mL DPPH ethanol solution with 2 mL of sample ethanol solution at a 1:1 ratio (equal volumes of ethanol solution replacing the sample solution serve as the blank group, and equal volumes of ethanol solution replacing the DPPH solution serve as the control group), and then incubate at 25°C in the dark for 20 min. Step b: After incubation, measure the absorbance at 320 nm using a UV-Vis spectrophotometer, and calculate the DPPH scavenging rate using the following formula:

[0118]

[0119] 2.2 Experimental Results

[0120] Employing DPPH free radical scavenging ability (IC) 50 The antioxidant capacity of FA-ES and FA is evaluated using a metric, with smaller values ​​indicating stronger free radical scavenging ability and stronger antioxidant activity. For example... Figure 4 As shown, the IC of FA-ES 50 The concentration was 3.69 μg / mL, and the IC50 of FA was... 50The concentration was 5.87 μg / mL. The experimental results showed that the DPPH radical scavenging ability of FA-ES was stronger than that of free FA.

[0121] 3. In vivo efficacy experiments of nematodes

[0122] 3.1 Transdermal absorption experiment in nematodes

[0123] Experimental methods:

[0124] Step a: Two days after synchronization, L4 stage Caenorhabditis elegans were washed with M9 buffer and collected into 10 mL EP tubes. The tubes were washed 2-3 times, allowed to settle naturally, and the supernatant was removed. Then, 0.5 mL of the worm solution was precisely transferred to FA-ES solution containing pyrene fluorescent probe. Step b: After staining for 20 min, the nematodes were washed 2-3 times with M9 buffer, photographed under an upright fluorescence microscope, and the fluorescence intensity of the nematodes was measured using Image J.

[0125] 3.2 Experimental Results

[0126] like Figure 5 and 6 As shown, the permeability of FA-ES to the stratum corneum of nematode skin can be tracked and measured by fluorescence microscopy. The relative fluorescence intensity of FA-ES to FA to permeate the stratum corneum of nematode skin is increased by 113.14%. The experimental results show that after FA-ES is made, it can enhance the permeability of FA to the stratum corneum of skin.

[0127] 3.3 Nematode lifespan experiment

[0128] Experimental methods:

[0129] Step a: Two days after synchronization, select 30 L4-stage Caenorhabditis elegans nematodes and transfer them to nematode culture dishes coated with blank solution, drug solutions (FA-ES solution and FA solution), and 10 μg / mL vitamin C solution. Incubate in a nematode culture incubator. Record the nematode survival status daily. From day one to day five, transfer the nematodes to a new drug-treated culture dish daily. After day five, transfer the nematodes to a new drug-treated culture dish every other day until all nematodes in the culture dish die.

[0130] 3.4 Experimental Results

[0131] like Figure 7As shown, the average lifespan of the control group was 7.52±0.31 days. When the FA-ES and FA concentrations were 10 μg / mL, the average lifespans of the nematodes were 8.76±0.87 days and 8.52±0.18 days, respectively, with a relative lifespan extension of 116.40% and 113.29% compared to the control group. When the FA concentration was 5 μg / mL, the average lifespans of the nematodes were 8.39±1.15 days and 8.33±0.53 days, respectively, with a relative lifespan extension of 111.52% and 110.78% compared to the control group. The experimental results indicate that FA-ES can prolong the lifespan of nematodes and has a good anti-aging effect.

[0132] 3.5 Nematode Lipofuscin Experiment

[0133] Experimental methods:

[0134] Step a: Two days after synchronization, L4 stage *C. elegans* were picked and placed onto NGM culture dishes coated with blank solution, drug solutions (FA-ES solution and FA solution), and 10 μg / mL vitamin C solution. The dishes were then placed in a nematode incubator and cultured. The day of picking was recorded as day 0. Step b: Each day, the nematodes were picked and placed onto a new drug-treated culture dish. After 10 days of culture, the nematodes were anesthetized with 2% sodium azide and then picked onto a slide containing 2% agarose. The slides were photographed under a fluorescence microscope, and the fluorescence intensity was measured using ImageJ.

[0135] 3.6 Experimental Results

[0136] As the body ages, cellular metabolism is disrupted, leading to the production of large amounts of free radicals, which induce the production and accumulation of lipofuscin. For example... Figure 8 and 9 As shown, when the concentrations of FA-ES and FA were 10 μg / mL, the relative fluorescence intensities of lipofuscin in nematodes were 67.32% and 74.07%, respectively. When the concentrations of FA-ES and FA were 5 μg / mL, the relative fluorescence intensities of lipofuscin in nematodes were 78.31% and 82.51%, respectively. The experimental results indicate that FA-ES can inhibit the growth of lipofuscin in nematodes and has a good anti-aging effect.

[0137] 3.7 Nematode Oxidative Stress Experiment

[0138] Experimental methods:

[0139] Step a: Two days after synchronization, 20 L4-stage *C. elegans* nematodes were picked and placed onto NGM culture dishes coated with blank solution, drug solutions (FA-ES solution and FA solution), and 10 μg / mL vitamin C solution. The dishes were then placed in a nematode incubator and cultured. The day of picking was recorded as day 0. Step b: After two days of culture, the nematodes were picked onto NGM culture dishes containing 2.2 mmol / L H2O2. The survival rate of the nematodes was recorded every hour until all the nematodes in the culture dishes died.

[0140] 3.8 Experimental Results

[0141] like Figure 10 As shown, the average lifespan of nematodes in the control group was 1.48 ± 0.08 hours. When the FA-ES and FA concentrations were 10 μg / mL, the average lifespans of nematodes were 2.03 ± 0.16 hours and 1.73 ± 0.08 hours, respectively, representing a lifespan extension of 137.08% and 116.85% relative to the control group. When the FA concentration was 5 μg / mL, the average lifespans of nematodes were 1.87 ± 0.10 hours and 1.63 ± 0.18 hours, respectively, representing a lifespan extension of 125.84% and 110.11% relative to the control group. The experimental results indicate that FA-ES can prolong the average lifespan of nematodes under oxidative stress and has good antioxidant effects.

[0142] 3.9 Nematode Reproductive Capacity Experiment

[0143] Experimental methods:

[0144] Step a: Two days after synchronization, L4-stage *C. elegans* were picked and placed onto NGM culture dishes coated with blank solution, drug solutions (FA-ES solution and FA solution), and 10 μg / mL vitamin C solution. One nematode was picked from each dish. The nematodes were transferred to a new drug-treated culture dish daily until the nematodes stopped laying eggs. Step b: The number of progeny nematodes on each culture dish was recorded daily.

[0145] 3.10 Experimental Results

[0146] like Figure 11 As shown, the total number of eggs laid by nematodes in the control group was 158.33±4.16. When the concentration of FA-ES and FA was 10 μg / mL, the total number of eggs laid by nematodes was 211.67±16.26 and 214.33±14.19, respectively. When the concentration was 5 μg / mL, the total number of eggs laid by nematodes was 206.00±18.68 and 197.00±14.73, respectively. The experimental results show that the total number of eggs laid in each group was higher than that in the control group. Therefore, FA-ES is non-toxic to the reproductive system of nematodes and can improve the reproductive capacity of nematodes.

[0147] 3.11 Nematode High Temperature Stress Experiment

[0148] Experimental methods:

[0149] Step a: Two days after synchronization, 20 L4-stage *C. elegans* nematodes were picked and placed onto NGM culture dishes coated with blank solution, drug solutions (FA-ES solution and FA solution), and 10 μg / mL vitamin C solution. These dishes were then placed in a nematode incubator. The day of picking was recorded as day 0. Step b: After two days of incubation, the nematodes were picked onto NGM culture dishes and placed in a 35°C incubator. The survival rate of the nematodes was recorded every hour until all nematodes in the culture dish died.

[0150] 3.12 Experimental Results

[0151] like Figure 12 As shown, the average lifespan of nematodes in the control group was 3.97 ± 0.08 hours. When the FA-ES and FA concentrations were 10 μg / mL, the average lifespans of nematodes were 5.23 ± 0.03 hours and 4.77 ± 0.08 hours, respectively, representing a lifespan extension of 137.08% and 116.85% relative to the control group. When the FA concentration was 5 μg / mL, the average lifespans of nematodes were 4.98 ± 0.24 hours and 4.57 ± 0.20 hours, respectively, representing a lifespan extension of 125.84% and 110.11% relative to the control group. The experimental results indicate that FA-ES can prolong the average lifespan of nematodes under high-temperature conditions and has a good anti-aging effect.

[0152] 4. Experiment on the efficacy of zebrafish in vivo

[0153] 4.1 Zebrafish Embryo Stimulation Experiment

[0154] Experimental methods:

[0155] Step a: Take zebrafish embryos 6 hours after fertilization (6 hpf) and transfer them to a six-well plate containing 5 mL of blank solution, drug solution (FA-ES solution and FA solution) and 10 μg / mL vitamin C solution, with 20 embryos per well. The incubation volume of the drug solution is 5 mL. Step b: Record the survival status of zebrafish and change the drug solution every 12 hours, and observe continuously for 72 hours.

[0156] 4.2 Experimental Results

[0157] like Figure 13As shown, 72 hours after administration to zebrafish, the survival rates of zebrafish embryos treated with FA-ES and FA were 98.33±2.89% and 86.67±5.77%, respectively, at a concentration of 10 μg / mL. At a concentration of 5 μg / mL, the survival rates of zebrafish embryos treated with FA-ES and FA were both 96.67±2.89%. The experimental results indicate that preparing FA-ES can effectively reduce the irritant effect of FA on zebrafish embryos and improve the safety of the drug.

[0158] 4.3 Zebrafish Moisturizing Experiment

[0159] Experimental methods:

[0160] Step a: Zebrafish larvae at 72 hpf post-fertilization were placed on a glass slide and laid flat for photographing under a stereomicroscope. The larvae were then transferred to 96-well plates and divided into three groups: a model group (100 μL 4% sodium chloride solution + 100 μL embryo culture water), a sample group (100 μL 4% sodium chloride solution + 100 μL drug solution), and a positive control group (100 μL 4% sodium chloride solution + 100 μL 20 μg / mL sodium hyaluronate solution). Step b: After drug administration, the larvae were incubated in a zebrafish incubator for 15 min. Then, the larvae were placed on a glass slide and laid flat for photographing under a stereomicroscope. The degree of tail wrinkling was measured using ImageJ.

[0161] 4.4 Experimental Results

[0162] like Figure 14 and 15 As shown, at a drug concentration of 10 μg / mL, the moisturizing rates of FA-ES and FA were 94.19±0.96% and 90.73±5.11%, respectively; at a drug concentration of 5 μg / mL, the moisturizing rates of FA-ES and FA were 92.20±3.86% and 90.09±2.83%, respectively. The experimental results indicate that both FA-ES and FA can alleviate the dehydration and shrinkage of the tail of zebrafish juveniles under high osmotic pressure, demonstrating good moisturizing effects.

[0163] 4.5 Zebrafish Oxidative Stress Experiment

[0164] Experimental methods:

[0165] Step a: Zebrafish larvae at 6 hpf post-fertilization were transferred to 6-well plates and administered the following drugs: model group (2.5 mL 7 mmol / L H2O2 + 2.5 mL embryo culture water), sample group (2.5 mL 7 mmol / L H2O2 + 2.5 mL drug solution), and positive control group (2.5 mL 7 mmol / L H2O2 + 2.5 mL 10 μg / mL vitamin C solution). Step b: After drug administration, the zebrafish were placed in a zebrafish incubator and cultured in the dark. The survival rate of the zebrafish embryos was recorded every 12 hours, and the drug solution was changed accordingly. Observation continued for 72 hours.

[0166] 4.6 Experimental Results

[0167] like Figure 16 As shown, at a drug concentration of 5 μg / mL, the embryo mortality rates of zebrafish embryos treated with FA-ES and FA after 72 h were 58.33±7.63% and 73.33±10.40%, respectively. At a drug concentration of 10 μg / mL, the embryo mortality rates of zebrafish embryos treated with FA-ES were 41.67±7.63%, and those treated with FA were 66.67±7.63%. The experimental results indicate that both FA-ES and FA have a good protective effect against H2O2 oxidative stress in zebrafish embryos.

[0168] 4.7 Zebrafish Repair Experiment

[0169] Experimental methods:

[0170] Step a: Zebrafish larvae at 72 hpf after fertilization were placed on a glass slide and briefly anesthetized with 0.08% tricaine. The caudal fin was removed along the end of the spine using a stereomicroscope. The zebrafish larvae were immediately transferred to embryo culture water for resuscitation. After resuscitation, they were transferred to 96-well plates. The drug administration groups were: model group (200 μL embryo culture water) and sample group (200 μL FA-ES solution and FA solution). Step b: After drug administration, the zebrafish were placed in a zebrafish incubator and cultured. The drug solution was changed every 12 hours. Two days later, the zebrafish larvae were placed on a glass slide and laid flat. The images were taken under a stereomicroscope, and the regenerated caudal fin length of the zebrafish larvae was measured using ImageJ.

[0171] 4.8 Experimental Results

[0172] like Figure 17 and 18As shown, the regenerated tail fin length of zebrafish juveniles in the control group was 36.67±10.11 μm. When the FA-ES and FA concentrations were 10 μg / mL, the regenerated tail fin lengths were 89.33±10.02 μm and 68.00±12.29 μm, respectively. At a concentration of 5 μg / mL, the regenerated lengths were 70.00±21.28 μm and 53.67±13.58 μm, respectively. The experimental results indicate that both FA-ES and FA have a certain regenerative effect on zebrafish.

[0173] 4.9 β-Galactosidase Activity Assay

[0174] Experimental methods:

[0175] Step a: Zebrafish larvae at 6 hpf post-fertilization were transferred to 6-well plates. The treatment groups were: a blank group (5 mL embryo culture water) and a sample group (5 mL FA-ES solution and FA solution). After treatment, the larvae were placed in a zebrafish incubator, with the drug solution changed every 12 hours. After 72 hpf of treatment, the larvae were washed 2-3 times with embryo culture water, then transferred to 96-well plates. 100 μL of fixative was added, and the plates were fixed at 4°C for two days. The larvae were then washed 2-3 times with embryo culture water, and 100 μL of staining solution was added. The plates were incubated at 37°C for 12 hours. Step b: After incubation, the larvae were placed on a glass slide and laid flat. Images were taken under a stereomicroscope, and the staining intensity of β-galactosidase in the larvae was measured using ImageJ.

[0176] 4.10 Experimental Results

[0177] like Figure 19 and 20 As shown, when the FA-ES and FA concentrations were 10 μg / mL, the β-galactosidase activities in juvenile zebrafish were 69.55±11.46% and 79.69±8.86%, respectively; when the concentration was 5 μg / mL, the β-galactosidase activities in juvenile zebrafish were 83.32±11.32% and 85.54±5.65%, respectively. The experimental results indicate that both FA-ES and FA have certain anti-aging effects on zebrafish.

[0178] 4.11 Zebrafish Melanin Inhibition Experiment

[0179] 4.11.1 Experiment on melanin area in zebrafish

[0180] Experimental methods:

[0181] Step a: Zebrafish larvae at 6 hpf post-fertilization were transferred to 6-well plates. The treatment groups were: a blank group (5 mL embryo culture water), a sample group (5 mL FA-ES solution and FA solution), and a positive control group (5 mL 10 μg / mL α-arbutin solution). After treatment, the larvae were placed in a zebrafish incubator in the dark. Step b: The drug solution was changed every 12 hours. After 72 hpf of treatment, the zebrafish larvae were placed on a glass slide and laid flat for photographing under a stereomicroscope.

[0182] 4.11.2 Experimental Results

[0183] like Figure 21 As shown, when FA-ES and FA were administered at concentrations of 5 and 10 μg / mL, respectively, zebrafish juveniles showed less melanin synthesis and lighter body color, while the control group showed darker body color and more melanin synthesis. The experimental results indicate that both FA-ES and FA inhibit melanin synthesis in zebrafish juveniles.

[0184] 4.11.3 Zebrafish Melanin Synthesis Experiment

[0185] Experimental methods:

[0186] Step a: Zebrafish larvae at 6 hpf post-fertilization were transferred to 6-well plates. The treatment groups were: blank group (5 mL embryo culture water), sample group (5 mL drug solution), and positive control group (5 mL 10 μg / mL α-arbutin solution). After administration, the zebrafish were placed in a zebrafish incubator in the dark. The drug solution was changed every 12 h. After 72 hpf of administration, the zebrafish were washed 2-3 times with embryo culture water. Thirty zebrafish larvae were transferred to 1.5 mL EP tubes, centrifuged at 5000 rpm for 5 min, and the supernatant was removed. 150 μL of 5 mg / mL sodium deoxycholate solution was added, and the mixture was homogenized using a tissue homogenizer. Then, 150 μL of 40 mg / mL NaOH solution was added to the EP tubes, and the mixture was heated in a boiling water bath for 15 min. After heating, the mixture was vortexed to fully dissolve the melanin in the EP tubes. Step b: Finally, take 100 μL of the solution and place it in a 96-well plate. Measure the absorbance at 405 nm using a microplate reader. Calculate the melanin content of zebrafish juveniles using the following formula:

[0187]

[0188] 4.11.4 Experimental Results

[0189] like Figure 22As shown, when the concentrations of FA-ES and FA were 10 μg / mL, the melanin contents of zebrafish juveniles were 85.17±1.10% and 90.31±0.89%, respectively; when the concentration was 5 μg / mL, the melanin contents of zebrafish juveniles were 90.76±0.57% and 93.38±0.51%, respectively. The experimental results indicate that both FA-ES and FA have an inhibitory effect on melanin synthesis in zebrafish.

[0190] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Ferulic acid ethosomes having multiple anti-aging efficacy, characterized in that, The ferulic acid alcohol microsomes comprise 80-100 parts of soybean lecithin, 5 parts of astragalus glycoside, 5 parts of ceramide and 10-15 parts of ferulic acid by mass fraction.

2. The ferulic acid ethanolsome according to claim 1, characterized in that, The mass ratio of the soybean lecithin, astragalus glycoside, ceramide and ferulic acid is 18:1:1:

2.

3. The method for preparing the ferulic acid alcohol liposome according to any one of claims 1 or 2, characterized in that, The method comprises the following steps: A. mixing the formula amount of soybean lecithin, astragalus glycoside, ceramide and ferulic acid to obtain a mixture, adding anhydrous ethanol to the mixture, stirring until dissolved to obtain an organic phase; B. injecting the organic phase obtained in step A into an aqueous phase to hydrate to obtain a solution; C. filtering the solution obtained in step B to obtain the ferulic acid alcohol microsomes.

4. The production method according to claim 3, characterized by, The stirring mode in step A is constant temperature stirring, and the constant temperature is 45℃.

5. The preparation method according to claim 3, characterized in that, The mass-volume ratio of the anhydrous ethanol to the mixture in step A is 100-120 mg:1.5 mL.

6. The preparation method according to claim 3, characterized in that, The rotation speed of the hydration in step B is 800 rpm, the hydration temperature is 45℃, and the hydration time is 20 min.

7. The preparation method according to claim 3, characterized in that, The volume ratio of the aqueous phase to the organic phase in step B is 2-3:

1.

8. The preparation method according to claim 3, characterized in that, The pore size of the filtration in step C is 0.22 μm, and the number of filtration is 3 times.

9. Use of the ferulic acid alcohol microsomes of any one of claims 1 or 2 or obtained by the preparation method of any one of claims 3-8 in the preparation of a product with multiple anti-aging effects.

10. A product having multiple anti-aging efficacy, characterized in that, The product comprises the ferulic acid alcohol microsomes of any one of claims 1 or 2.

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