Ferulic acid ethosome with multiple anti-aging effects as well as preparation method, application and product of ferulic acid ethosome

Abstract

The invention relates to the technical field of cosmetics, in particular to ferulic acid ethosome with multiple anti-aging effects, a preparation method and application of the ferulic acid ethosome and a product of the ferulic acid ethosome. The ferulic acid ethosome is prepared from the following components in parts by mass: 80 to 100 parts of soybean lecithin, 5 parts of astragaloside, 5 parts of ceramide and 10 to 15 parts of ferulic acid. The preparation method of the ferulic acid ethosome comprises the following steps: A, mixing soybean lecithin, astragaloside, ceramide and ferulic acid according to the formula dosage to obtain a mixed material, adding absolute ethyl alcohol into the mixed material, and stirring until the absolute ethyl alcohol is dissolved to obtain an organic phase; b, injecting the organic phase obtained in the step A into water, and hydrating to obtain a solution; and C, filtering the solution obtained in the step B to obtain the ferulic acid ethosome. According to the ferulic acid ethosome disclosed by the invention, the risk of cardiovascular and cerebrovascular diseases of an organism is avoided, meanwhile, the carrier stability is also improved, and more skin care effects are brought.

Description

Technical Field

[0001] The invention relates to the field of cosmetics and skin care, and in particular to a ferulic acid ethosome with multiple anti-aging effects, a preparation method thereof, an application thereof and a product thereof. Background Art

[0002] The skin is the outermost protective organ of the human body and the first natural barrier of the human body to the outside world. It plays an important role in defending against the invasion of the external environment. The skin is divided into two layers: the epidermis and the dermis. The epidermis is on the surface of the skin and can be divided into two parts: the stratum corneum and the stratum germinativum. The stratum corneum is composed of mature keratinocytes and intercellular lipids. Among them, keratinocytes are "bricks" and intercellular lipids are "mortar". The two are closely arranged in a stable brick wall structure to ensure the integrity of the skin barrier. The intercellular lipids in the stratum corneum are composed of about 50% ceramide, 25% cholesterol, and 15% free fatty acids. They are important 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 abnormality and deficiency may lead to damage to the skin barrier function and cause skin problems.

[0003] Ferulic acid (FA) is a phenolic acid compound widely found in the plant kingdom. It is cross-linked with cell wall polysaccharides and lignin and is one of the main components of plant cell walls. Ferulic acid is an active ingredient in many foods, such as cereal foods such as wheat bran and rice bran; fruits such as citrus and bananas; vegetables such as eggplant and broccoli are all rich sources of ferulic acid for people. Ferulic acid has multiple anti-aging effects on the human body, including antioxidant, whitening, anti-inflammatory, anti-cancer, sunscreen, moisturizing and repair effects. Therefore, ferulic acid has a very wide application prospect in the cosmetics industry. However, due to its poor solubility and stability, human skin has low absorption and bioavailability. In addition, light, heat, oxidation and acidic environments can easily lead to a decrease in the efficacy of ferulic acid in various environments, resulting in limited application of ferulic acid. Emulsification systems such as nanoemulsions, liposomes, self-assembled micelles, etc. can be used to solve these problems. For example, Chinese patents CN115634167B and CN117159431A.

[0004] Since traditional emulsification technology often only stays in the stratum corneum of the skin and it is difficult to deliver active ingredients to the deep layers of the skin, Touitou et al. developed a new carrier composed of phospholipids, ethanol and water, which they defined as ethosomers (ES). Ethosomes are liposomes with 20%-50% (w / w) ethanol added, and they also have the ability to encapsulate hydrophilic and hydrophobic compounds.

[0005] For example, Deng Mengjie et al. published an article titled "Preparation, Characterization and In Vitro Transdermal Absorption of Ferulic Acid Nanoparticles" in the journal "Daily Chemical Industry (Chinese and English)" in 2023. The article studied the transdermal delivery and antioxidant properties of ferulic acid nanoparticles prepared by microfluidization technology. First, the response surface methodology was used to study the effects of lecithin and edge activators (sucrose palmitate, Tween-80) on the encapsulation efficiency of ferulic acid nanoparticles. 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 a small particle size and uniform distribution ((104.5±0.7) nm), a high encapsulation efficiency (88.8%±2.6%), and high stability (stable storage at 25°C for 60 days). Secondly, the antioxidant properties and transdermal delivery properties of ferulic acid nano-alcoholsomes were evaluated by DPPH free radical scavenging experiment and Franz diffusion cell in vitro transdermal experiment, respectively. The study found that the prepared ferulic acid nano-alcoholsomes have uniform particle size, high encapsulation rate, good stability, high antioxidant and good transdermal absorption performance, and are expected to be used in transdermal delivery systems of phenolic acid natural active ingredients, as well as cosmetics with antioxidant, anti-aging and whitening effects. Although the encapsulation rate and stability of ferulic acid alcosomes prepared in this article are very good, the preparation process is complicated. However, a major disadvantage of alcosomes is that the fluidity of the phospholipid bilayer is relatively high. This article did not make a specific study on this, so it is necessary to find an ingredient that can reduce the fluidity of the alcosomes.

[0006] Secondly, cholesterol acts as a stabilizer for the fluidity of the molecular membrane of the phospholipid bilayer structure, and 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, so there is an urgent need to find an ingredient that can replace cholesterol as a stabilizer and also have anti-aging effects.

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

[0008] Based on the deficiencies of the prior art, the present invention aims to provide a ferulic acid ethosome with simple preparation method, low fluidity, good stability, high transdermal absorption rate, whitening and multiple anti-aging effects, and its preparation method, application and product. The ferulic acid ethosome comprises: soybean lecithin, astragaloside IV, ceramide and ferulic acid. Experiments have shown that the ferulic acid ethosome has not only good stability and uniform particle size, but also high transdermal absorption rate, can inhibit melanin synthesis, and has significant whitening and anti-oxidation effects.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

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

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

[0012] In a second aspect, the present invention also provides a method for preparing the above-mentioned ferulic acid ethosomes having multiple anti-aging effects, characterized in that it comprises the following steps:

[0013] A. Mixing the soybean lecithin, astragaloside IV, ceramide and ferulic acid in the formula to obtain a mixture, adding a solvent to the mixture, stirring until dissolved, and obtaining an organic phase;

[0014] B. injecting the organic phase obtained in step A into the aqueous phase for hydration to obtain a solution;

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

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

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

[0018] More preferably, the mass volume ratio of anhydrous ethanol to the mixed material 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 a syringe.

[0023] Preferably, the filter used for filtration in step C is an organic microporous filter membrane, the pore size of the filter is 0.22 μm, and the filtration is performed 3 times.

[0024] In a third aspect, the present invention also provides the use of the above-mentioned ferulic acid ethosomes with multiple anti-aging effects in the preparation of products with multiple anti-aging effects.

[0025] In a fourth aspect, the present invention also provides a product having multiple anti-aging effects of the ferulic acid ethosomes having multiple anti-aging effects. Preferably, the product is in the form of an emulsion.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The ferulic acid in the present invention has better solubility and stability, and can deliver active ingredients to the deep layers of the skin, has strong flexibility, and has high transdermal absorption efficiency.

[0028] (2) A major disadvantage of ethosomes is that the fluidity of the phospholipid bilayer is relatively high. The present invention adds ceramide as a membrane modifier to reduce the fluidity of ethosomes, improve their stability and enhance their transdermal permeability to the skin.

[0029] (3) The present invention utilizes astragaloside IV to replace cholesterol, thereby preventing cardiovascular and cerebrovascular diseases and bringing more skin care effects to the ethanol body.

[0030] (4) The present invention adds astragaloside IV and ceramide as membrane modifiers to ethosomes for encapsulating ferulic acid, thereby achieving multiple anti-aging effects. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0032] Figure 2 The particle size and dispersion index (PDI) of FA-ES after being placed at different temperatures for 30 days;

[0033] Figure 3 Zeta potential at different temperatures for 30 days;

[0034] Figure 4 This is the DPPH free radical scavenging ability diagram;

[0035] Figure 5 This is a pyrene fluorescence microscopy image of nematodes;

[0036] Figure 6 This is a graph of the skin permeability of nematodes;

[0037] Figure 7 This is a graph of the life span of nematodes;

[0038] Figure 8 This is a fluorescence microscopy image of lipofuscin in nematodes;

[0039] Fig. 9is a graph of lipofuscin levels in C. elegans;

[0040] Fig.10 is a diagram of oxidative stress in C. elegans;

[0041] Fig.11 This is a graph of the total egg production of nematodes within 7 days;

[0042] Fig.12 This is a diagram of high temperature stress in nematodes;

[0043] Fig.13 Embryonic stimulation diagram for zebrafish;

[0044] Fig.14 and 15 This is the moisture retention rate diagram of zebrafish;

[0045] Fig.16 is a diagram of oxidative stress in zebrafish;

[0046] Fig.17 and 18 This is a restoration diagram for zebrafish;

[0047] Fig.19 and 20 This is a diagram of the β-galactosidase staining degree of zebrafish;

[0048] Fig.21 and 22 This is a graph showing the melanin content in zebrafish. DETAILED DESCRIPTION

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

[0050] Example 1

[0051] The ferulic acid ethosome specifically comprises, by weight, 90 parts of soy lecithin, 5 parts of astragaloside IV, 5 parts of ceramide and 10 parts of ferulic acid.

[0052] The preparation method of ferulic acid ethosomes comprises the following steps:

[0053] A: Ethanol injection method was used to prepare ethosomes. First, 90 mg of soybean lecithin, 5 mg of astragaloside IV, 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 an organic phase.

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

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

[0056] Example 2

[0057] The ferulic acid ethosome specifically comprises, by weight, 80 parts of soybean lecithin, 5 parts of astragaloside IV, 5 parts of ceramide and 10 parts of ferulic acid.

[0058] The preparation method of ferulic acid ethosomes comprises the following steps:

[0059] A: Ethanol injection method was used to prepare ethosomes. First, 80 mg of soybean lecithin, 5 mg of astragaloside IV, 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 an organic phase.

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

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

[0062] Example 3

[0063] The ferulic acid ethosome specifically comprises, by weight, 100 parts of soybean lecithin, 5 parts of astragaloside IV, 5 parts of ceramide and 10 parts of ferulic acid.

[0064] The preparation method of ferulic acid ethosomes comprises the following steps:

[0065] A: Ethanol injection method was used to prepare ethosomes. First, 100 mg of soybean lecithin, 5 mg of astragaloside IV, 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 an organic phase.

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

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

[0068] Example 4

[0069] The ferulic acid ethosome specifically comprises, by weight, 90 parts of soy lecithin, 5 parts of astragaloside IV, 5 parts of ceramide and 15 parts of ferulic acid.

[0070] The preparation method of ferulic acid ethosomes comprises the following steps:

[0071] A: Ethanol injection method was used to prepare ethosomes. First, 90 mg of soybean lecithin, 5 mg of astragaloside IV, 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 an organic phase.

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

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

[0074] The preparation method is consistent with that in Example 1.

[0075] Example 5

[0076] The composition and mass fraction of ferulic acid ethosomes are consistent with those in Example 1.

[0077] The preparation method of ferulic acid ethosomes comprises the following steps:

[0078] A: Ethanol injection method was used to prepare ethosomes. First, 90 mg of soybean lecithin, 5 mg of astragaloside IV, 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 an organic phase.

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

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

[0081] Example 6

[0082] The composition and mass fraction of ferulic acid ethosomes are consistent with those in Example 1.

[0083] The preparation method of ferulic acid ethosomes comprises the following steps:

[0084] A: Ethanol injection method was used to prepare ethosomes. First, 90 mg of soybean lecithin, 5 mg of astragaloside IV, 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 an organic phase.

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

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

[0087] Comparative Example 1

[0088] The ferulic acid ethosome specifically comprises, by weight: 90 parts of soybean lecithin, 6.25 parts of astragaloside IV, 3.75 parts of ceramide and 10 parts of ferulic acid.

[0089] The preparation method of ferulic acid ethosomes comprises the following steps:

[0090] A: Ethanol injection method was used to prepare ethosomes. First, 90 mg of soybean lecithin, 6.25 mg of astragaloside IV, 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 an organic phase.

[0091] The remaining steps are consistent with those in Example 1.

[0092] Comparative Example 2

[0093] The ferulic acid ethosome specifically comprises, by weight: 90 parts of soybean lecithin, 3.75 parts of astragaloside IV, 6.25 parts of ceramide and 10 parts of ferulic acid.

[0094] The preparation method of ferulic acid ethosomes comprises the following steps:

[0095] A: Ethanol injection method was used to prepare ethosomes. First, 90 mg of soybean lecithin, 3.75 mg of astragaloside IV, 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 an organic phase.

[0096] The other steps are consistent with those in Example 1.

[0097] Comparative Example 3

[0098] The ferulic acid ethosome specifically comprises, by weight, 90 parts of soybean lecithin, 5 parts of astragaloside IV, 5 parts of ceramide and 20 parts of ferulic acid.

[0099] The preparation method of ferulic acid ethosomes comprises the following steps:

[0100] A: Ethanol injection method was used to prepare ethosomes. First, 90 mg of soybean lecithin, 5 mg of astragaloside IV, 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 an organic phase.

[0101] The other steps are consistent with those in Example 1.

[0102] Effect Experiment

[0103] 1. Characterization of Ferulic Acid Ethosomes

[0104] 1.1 Experimental methods

[0105] Determination of encapsulation efficiency: Step a: Separate FA-ES and FA by ultrafiltration centrifugation. Take 0.5 mL of the newly prepared FA-ES, place it in a 10 mL volumetric flask, add an appropriate amount of anhydrous ethanol, perform ultrasonic demulsification for 15 minutes, and then add anhydrous ethanol to make up the volume. After filtering through a 0.22 μm microporous filter membrane, use a UV-visible spectrophotometer to determine the total FA content (W t Step b: Accurately pipette 0.5 mL of the same FA-ES into an ultrafiltration centrifuge tube with a cutoff molecular weight of 10 kDa, centrifuge at 4°C, 12000 rpm for 15 min, collect the filtrate, and measure the free FA content (W f ), and the encapsulation efficiency was calculated according to the following formula:

[0106]

[0107] Determination of particle size, PDI and Zeta potential: Step a: Take the newly prepared FA-ES solution and dilute it with pure water. Step b: Use Malvern particle size potential analyzer 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 three times in parallel.

[0108] Determination of storage stability: Step a: Take the newly prepared FA-ES solution, place it in a vial, and place it at 4°C, 25°C and 37°C for 30 days. Step b: Use a Malvern particle size analyzer to measure the particle size, PDI and Zeta potential of FA-ES at a scattering angle of 90° and a temperature of 25°C 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 influence of various factors on the alcohol body was investigated with particle size, PDI, Zeta potential and encapsulation efficiency as indicators, and the process was optimized. Figure 1 , Figure 2 , Figure 3 As shown, after the FA-ES of Example 1 was placed at 4°C, 25°C and 37°C for 30 days, the particle size, PDI and Zeta potential stability of the FA-ES of the embodiment and the comparative example were good, while the encapsulation efficiency of the comparative example was not as good as that of the embodiment, indicating that the mass ratio of ceramide and astragaloside IV was 1:1 and the encapsulation efficiency was better when the components contained in the alcohol body were within the scope limited by the present invention. The optimal preparation conditions were finally determined to be: 90 mg of soybean lecithin, 5 mg of astragaloside IV, 5 mg of ceramide, 10 mg of ferulic acid, 30% of anhydrous ethanol volume fraction, 45°C of hydration temperature, 800 rpm of hydration speed, 20 min of hydration time, 52.87 ± 1.56% of alcohol body encapsulation efficiency, 147.8 ± 0.72 nm of particle size, 0.12 ± 0.01 of PDI, and -32.3 ± 0.90 mv of Zeta potential.

[0111] Table 1

[0112]

[0113]

[0114] 2. In vitro antioxidant test

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

[0116] 2.1 DPPH free radical scavenging experiment method

[0117] Step a: Take 2mL of 50μg / mL DPPH ethanol solution and mix it with 2mL of sample ethanol solution in a ratio of 1:1 (the blank group is replaced by the equal volume of ethanol solution instead of the sample solution, and the control group is replaced by the equal volume of ethanol solution instead of the DPPH solution), and then incubate at 25℃ in a dark environment for 20min. Step b: After the incubation is completed, the absorbance is measured at a wavelength of 320nm using a UV-visible spectrophotometer, and the DPPH clearance rate is calculated according to the following formula:

[0118]

[0119] 2.2 Experimental Results

[0120] The DPPH free radical scavenging capacity (IC 50 ) was used to evaluate the antioxidant capacity of FA-ES and FA. The smaller the value, the stronger the free radical scavenging ability and the stronger the antioxidant activity. Figure 4 As shown, FA-ES IC 50 The IC value of FA is 3.69 μg / mL. 50The experimental results showed that the DPPH free radical scavenging ability of FA-ES was stronger than that of free FA.

[0121] 3. In vivo efficacy experiments in nematodes

[0122] 3.1 In vivo transdermal absorption experiment in nematodes

[0123] Experimental methods:

[0124] Step a: The L4 stage Caenorhabditis elegans two days after synchronization were rinsed with M9 buffer and collected into a 10 mL EP tube, washed 2-3 times, and the supernatant was aspirated after natural sedimentation. Then, 0.5 mL of the worm liquid was accurately transferred to the FA-ES solution containing the pyrene fluorescent probe. Step b: After staining for 20 minutes, 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 penetration ability of FA-ES into the stratum corneum of the nematode skin can be tracked and measured by fluorescence microscopy. The relative fluorescence intensity increase ratio of FA-ES to FA in the transdermal penetration of the nematode skin is 113.14%. The experimental results show that after FA-ES is made, the transdermal penetration ability of FA into the stratum corneum of the skin can be enhanced.

[0127] 3.3 Nematode lifespan experiment

[0128] Experimental methods:

[0129] Step a: Pick 30 L4-stage Caenorhabditis elegans two days after synchronization and place them on nematode growth culture dishes coated with blank solution, drug solution (FA-ES solution and FA solution) and 10 μg / mL vitamin C solution, and culture them in a nematode incubator. The day of picking is recorded as day 0. Step b: Record the survival of nematodes every day, transfer the nematodes to new drug-administered culture dishes every day from the first to the fifth day, and transfer the nematodes to new drug-administered culture dishes every other day after the fifth day until all the nematodes in the culture dishes die.

[0130] 3.4 Experimental Results

[0131] like Figure 7As shown, the average lifespan of the blank group was 7.52±0.31days. When the dosage concentration of FA-ES and FA was 10μg / mL, the average lifespan of the nematodes was 8.76±0.87days and 8.52±0.18days, respectively, which extended the lifespan by 116.40% and 113.29% relative to the blank; when the dosage concentration was 5μg / mL, the average lifespan of the nematodes was 8.39±1.15days and 8.33±0.53days, respectively, which extended the lifespan by 111.52% and 110.78% relative to the blank. The experimental results show that FA-ES can prolong the lifespan of nematodes and has good anti-aging effect.

[0132] 3.5 Nematode lipofuscin experiment

[0133] Experimental methods:

[0134] Step a: Pick the L4 stage Caenorhabditis elegans two days after synchronization onto NGM culture dishes coated with blank solution, drug solution (FA-ES solution and FA solution) and 10 μg / mL vitamin C solution, and culture them in a nematode incubator. The day of picking is recorded as day 0. Step b: Pick the nematodes onto a new drug-treated culture dish every day. After 10 days of culture, the nematodes are anesthetized with 2% sodium azide, picked onto a slide containing 2% agarose, and photographed under an upright fluorescence microscope. Image J is used to measure the fluorescence intensity of the nematodes.

[0135] 3.6 Experimental Results

[0136] As the body's aging level increases, cell metabolism is disrupted, which will produce a large number of free radicals and induce the production and accumulation of lipofuscin. Figure 8 and 9 As shown, when the dosage concentration of FA-ES and FA was 10 μg / mL, the relative fluorescence intensity of lipofuscin in nematodes was 67.32% and 74.07%, respectively. When the dosage concentration of FA-ES and FA was 5 μg / mL, the relative fluorescence intensity of lipofuscin in nematodes was 78.31% and 82.51%, respectively. The experimental results show that FA-ES can inhibit the growth of lipofuscin in nematodes and has good anti-aging effect.

[0137] 3.7 Nematode oxidative stress experiment

[0138] Experimental methods:

[0139] Step a: Pick 20 L4-stage Caenorhabditis elegans two days after synchronization onto NGM culture dishes coated with blank solution, drug solution (FA-ES solution and FA solution) and 10 μg / mL vitamin C solution, and culture them in a nematode incubator. The day of picking is recorded as day 0. Step b: After two days of culture, pick the nematodes onto NGM culture dishes containing 2.2 mmol / L H2O2, and record the survival of the nematodes every hour until all the nematodes in the culture dishes die.

[0140] 3.8 Experimental Results

[0141] like Fig.10 As shown, the average lifespan of nematodes in the blank group was 1.48±0.08hours. When the concentration of FA-ES and FA was 10μg / mL, the average lifespan of nematodes was 2.03±0.16hours and 1.73±0.08hours, respectively, which extended the lifespan by 137.08% and 116.85% relative to the blank group; when the concentration was 5μg / mL, the average lifespan of nematodes was 1.87±0.10hours and 1.63±0.18hours, respectively, which extended the lifespan by 125.84% and 110.11% relative to the blank group. The experimental results show that FA-ES can prolong the average lifespan of nematodes under oxidative stress and has good antioxidant effect.

[0142] 3.9 Nematode reproductive ability experiment

[0143] Experimental methods:

[0144] Step a: Pick L4 stage Caenorhabditis elegans two days after synchronization onto NGM culture dishes coated with blank solution, drug solution (FA-ES solution and FA solution) and 10 μg / mL vitamin C solution, pick one nematode from each dish, and transfer the nematodes to a new drug-treated culture dish every day until the nematodes stop laying eggs. Step b: Record the number of offspring of nematodes on each culture dish every day.

[0145] 3.10 Experimental Results

[0146] like Fig.11 As shown, the total number of nematode eggs laid in the blank group was 158.33±4.16, when the concentration of FA-ES and FA was 10μg / ml, the total number of nematode eggs laid were 211.67±16.26 and 214.33±14.19, respectively, when the concentration was 5μg / mL, the total number of nematode eggs laid were 206.00±18.68 and 197.00±14.73. The experimental results showed that the total egg laying amount of each group was higher than that of the blank group. It can be seen that 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: Pick 20 L4-stage Caenorhabditis elegans two days after synchronization and place them on NGM culture dishes coated with blank solution, drug solution (FA-ES solution and FA solution) and 10 μg / mL vitamin C solution, and place them in a nematode incubator for culture. The day of picking is recorded as day 0. Step b: After two days of culture, pick the nematodes onto the NGM culture dish and place them in a 35°C incubator for culture. Record the survival of the nematodes every hour until all the nematodes in the culture dish die.

[0150] 3.12 Experimental Results

[0151] like Fig.12 As shown, the average lifespan of nematodes in the blank group was 3.97±0.08hours. When the concentration of FA-ES and FA was 10μg / mL, the average lifespan of nematodes was 5.23±0.03hours and 4.77±0.08hours, respectively, which extended the lifespan by 137.08% and 116.85% relative to the blank group; when the concentration was 5μg / mL, the average lifespan of nematodes was 4.98±0.24hours and 4.57±0.20hours, respectively, which extended the lifespan by 125.84% and 110.11% relative to the blank group. The experimental results show that FA-ES can extend the average lifespan of nematodes in high temperature environment and has good anti-aging effect.

[0152] 4. Zebrafish in vivo efficacy experiment

[0153] 4.1 Zebrafish embryo stimulation experiment

[0154] Experimental methods:

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

[0156] 4.2 Experimental Results

[0157] like Fig.13As shown, 72 hours after zebrafish administration, when the concentration was 10 μg / mL, the zebrafish embryo survival rates of FA-ES and FA were 98.33±2.89% and 86.67±5.77%, respectively. When the concentration was 5 μg / mL, the zebrafish embryo survival rates of FA-ES and FA were both 96.67±2.89%. The experimental results showed that after FA-ES was prepared, the irritation of FA to zebrafish embryos could be effectively reduced, thereby improving the safety of the drug.

[0158] 4.3 Zebrafish moisturizing experiment

[0159] Experimental methods:

[0160] Step a: Take a zebrafish fry at 72 hpf after fertilization and lay it flat on a glass slide, and take pictures under a stereo microscope. Then transfer the zebrafish fry to a 96-well plate, and the drug administration groups are: model group (100 μL 4% sodium chloride solution + 100 μL embryo culture water), sample group (100 μL 4% sodium chloride solution + 100 μL drug solution) and positive control group (100 μL 4% sodium chloride solution + 100 μL 20 μg / mL sodium hyaluronate solution). Step b: After the drug administration is completed, place it in a zebrafish incubator for incubation for 15 minutes, then place the zebrafish fry on a glass slide to make it lie flat, take pictures under a stereo microscope, and use Image J to measure the degree of tail wrinkling of the zebrafish fry.

[0161] 4.4 Experimental Results

[0162] like Fig.14 and 15 As shown in the figure, when the dosage concentration was 10 μg / mL, the moisturizing rates of FA-ES and FA were 94.19±0.96% and 90.73±5.11%, respectively. When the dosage concentration was 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 showed that both FA-ES and FA could alleviate the dehydration and shrinkage of the tail of zebrafish larvae under high osmotic pressure, and had a good moisturizing effect.

[0163] 4.5 Zebrafish oxidative stress experiment

[0164] Experimental methods:

[0165] Step a: Take 6 hpf zebrafish larvae after fertilization and transfer them to a 6-well plate. The drug groups are: 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 the drug administration, place them in a zebrafish incubator in the dark, record the survival of zebrafish embryos and change the drug solution every 12 hours, and observe them continuously for 72 hours.

[0166] 4.6 Experimental Results

[0167] like Fig.16 As shown in the figure, when the dosage concentration was 5 μg / mL, the mortality of zebrafish embryos was 58.33±7.63% and 73.33±10.40% after 72 hours for FA-ES and FA, respectively. When the dosage concentration was 10 μg / mL, the mortality of zebrafish embryos was 41.67±7.63% for FA-ES and 66.67±7.63% for FA. The experimental results showed that both FA-ES and FA had good protective effects on zebrafish embryos under H2O2 oxidative stress.

[0168] 4.7 Zebrafish repair experiment

[0169] Experimental methods:

[0170] Step a: Take a zebrafish fry at 72 hpf after fertilization on a slide, anesthetize it briefly with 0.08% tricaine, and remove the tail fin along the end of the spine with the help of a stereo microscope. After the tail fin is removed, the zebrafish fry is immediately transferred to embryo culture water for recovery, and then transferred to a 96-well plate. The drug administration groups are: model group (200 μL embryo culture water) and sample group (200 μL FA-ES solution and FA solution). Step b: After the administration is completed, place it in a zebrafish incubator for culture, and change the drug solution every 12 hours. Two days later, place the zebrafish fry on a slide to make it lie flat, take pictures under a stereo microscope, and use Image J to measure the regenerated length of the zebrafish fry's tail fin.

[0171] 4.8 Experimental Results

[0172] like Fig.17 and 18As shown, the length of the caudal fin regeneration of zebrafish larvae in the blank group was 36.67±10.11μm, when the concentration of FA-ES and FA was 10μg / mL, the length of the caudal fin regeneration of zebrafish larvae was 89.33±10.02μm and 68.00±12.29μm, respectively, and when the concentration was 5μg / mL, the regeneration length was 70.00±21.28μm and 53.67±13.58μm, respectively. The experimental results show that both FA-ES and FA have a certain repair effect on zebrafish.

[0173] 4.9 β-Galactosidase activity assay

[0174] Experimental methods:

[0175] Step a: Take the zebrafish fry at 6 hpf after fertilization and transfer them to a 6-well plate. The drug groups are: blank group (5 mL embryo culture water) and sample group (5 mL FA-ES solution and FA solution). After the drug administration is completed, place them in a zebrafish incubator for culture, and change the drug solution every 12 hours. After the drug administration at 72 hpf, wash the zebrafish fry 2-3 times with embryo culture water, transfer the zebrafish fry to a 96-well plate, add 100 μL of fixative solution, fix for two days at 4°C, then wash with embryo culture water 2-3 times, add 100 μL of staining solution, and incubate at 37°C for 12 hours. Step b: After the incubation is completed, place the zebrafish fry on a slide to make it lie flat, take pictures under a stereo microscope, and use Image J to measure the staining degree of β-galactosidase in the zebrafish fry.

[0176] 4.10 Experimental Results

[0177] like Fig.19 and 20 As shown, when the concentration of FA-ES and FA was 10 μg / mL, the β-galactosidase activity of zebrafish larvae was 69.55±11.46% and 79.69±8.86%, respectively. When the concentration was 5 μg / mL, the β-galactosidase activity of zebrafish larvae was 83.32±11.32% and 85.54±5.65%, respectively. The experimental results show that both FA-ES and FA have certain anti-aging effects on zebrafish.

[0178] 4.11 Zebrafish melanin inhibition experiment

[0179] 4.11.1 Zebrafish melanin area experiment

[0180] Experimental methods:

[0181] Step a: Take 6hpf zebrafish fry after fertilization and transfer them to a 6-well plate. The drug groups are: blank group (5mL embryo culture water), sample group (5mL FA-ES solution and FA solution) and positive control group (5mL 10μg / mL α-arbutin solution). After the drug administration, place them in a zebrafish incubator in the dark. Step b: Change the drug solution every 12h. After 72hpf of drug administration, place the zebrafish fry on a slide to make it lie flat and take pictures under a stereo microscope.

[0182] 4.11.2 Experimental Results

[0183] like Fig.21 As shown in the figure, when the concentrations of FA-ES and FA were 5 and 10 μg / mL, the zebrafish larvae synthesized less melanin on their body surface and had a lighter body color, while the zebrafish larvae in the blank group had a darker body color and synthesized more melanin. The experimental results show that both FA-ES and FA have an inhibitory effect on melanin synthesis in zebrafish larvae.

[0184] 4.11.3 Zebrafish melanin synthesis experiment

[0185] Experimental methods:

[0186] Step a: Take zebrafish fry at 6 hpf after fertilization and transfer them to a 6-well plate. The drug groups are as follows: 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 the drug administration, place them in a zebrafish incubator in the dark and culture them. The drug solution is replaced every 12 hours. After the drug administration at 72 hpf, wash the zebrafish 2-3 times with embryo culture water, and draw 30 zebrafish fry into a 1.5 mL EP tube, centrifuge at 5000 rpm for 5 minutes, remove the supernatant, add 150 μL 5 mg / mL sodium deoxycholate solution, and use a tissue grinder to crush into a homogenate. Then, add 150 μL 40 mg / mL NaOH solution to the EP tube, heat it in a boiling water bath for 15 minutes, and vortex to mix after heating to fully dissolve the melanin in the EP tube. Step b: Finally, 100 μL of the solution was placed in a 96-well plate and the absorbance was measured at a wavelength of 405 nm using an ELISA reader. The melanin content of zebrafish fry was calculated according to the following formula:

[0187]

[0188] 4.11.4 Experimental Results

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

[0190] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A ferulic acid ethosome with multiple anti-aging effects, characterized in that: The ferulic acid ethosome comprises, by weight, 80-100 parts of soybean lecithin, 5 parts of astragaloside IV, 5 parts of ceramide and 10-15 parts of ferulic acid.

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

2.

3. The method for preparing ferulic acid ethosomes according to any one of claims 1 or 2, characterized in that: The steps include: A. Mixing the soybean lecithin, astragaloside IV, ceramide and ferulic acid in the formula to obtain a mixture, adding anhydrous ethanol to the mixture, stirring until dissolved, and obtaining an organic phase; B. injecting the organic phase obtained in step A into the aqueous phase for hydration to obtain a solution; C. Filter the solution obtained in step B to obtain the ferulic acid ethosome.

4. The preparation method according to claim 3, characterized in that: The stirring method in step A is constant temperature stirring, and the constant temperature is 45°C.

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

6. The preparation method according to claim 3, characterized in that: In step B, the hydration speed is 800 rpm, the hydration temperature is 45° C., 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 filtration is performed 3 times.

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

10. A product with multiple anti-aging effects, characterized in that: The invention comprises the ferulic acid ethosome according to any one of claims 1 or 2.

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