Nanoliposome with anti-cell oxidation and beta-galactosidase activity inhibition effects, preparation method and application thereof

Abstract

The invention discloses a nanoliposome with anti-cell oxidation and inhibitory effects on β-galactosidase activity, a preparation method and an application thereof, which belongs to the field of medical technology and solves the problem that the anti-aging active ingredients existing in the prior art are unstable and the effect is not ideal. The nanoliposome comprises components: 8-12wt% of cycloastragenol, 8-12wt% of ceramide, 15-25wt% of lecithin, 1-5wt% of proppant, 10-20wt% of solubilizer and 10-25wt% of stabilizer. The nanoliposome with anti-cell oxidation and inhibitory effects on β-galactosidase activity provided by the invention combines the mechanism and action site of aging, compounds ceramide with cycloastragenol, significantly activates the activity of telomerase, prolongs the length of telomeres, enhances tissue repair and regeneration process, and delays aging.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and more specifically to a nanoliposome having the functions of resisting cell oxidation and inhibiting the activity of beta-galactosidase, and a preparation method and application thereof. Background Art

[0002] The Chinese medicinal material Astragalus was first recorded in Shennong's Herbal Classic. Cycloastragenol (CAG) is a secondary metabolite of Astragalus, which is mainly obtained by hydrolysis of astragaloside, the effective pharmacologically active ingredient of Astragalus. Cycloastragenol is a cycloartane type in tetracyclic triterpenes, with poor water solubility and low oral bioavailability. The presence of cyclopropane groups in the structure leads to poor acid resistance, and it can be converted into astragalol under acidic conditions. Cycloastragenol can inhibit inflammatory aging by inhibiting the activation of inflammasome NLRP3; by inhibiting endoplasmic reticulum stress, it can maintain and reduce mitochondrial-dependent cell death. In 2010, Geron Company of the United States found in a research project to screen telomerase activators that cycloastragenol can activate the activity of telomerase, induce telomere extension, and has pharmacological effects such as anti-aging, anti-apoptosis, anti-fibrosis, and immunomodulation. With the development of society, my country has entered the stage of population aging, and the prevention of aging-related diseases has become the focus of people's attention. At present, cycloastragenol is the only reported telomerase activator among natural products and is one of the hot topics in anti-aging drug research.

[0003] Ceramide is a type of sphingolipid substance composed of sphingosine long-chain bases and fatty acids. It not only participates in the formation of cell membranes and maintains cell homeostasis, but also acts as a second messenger molecule in the sphingolipid pathway, playing an important role in the signal transduction process of cell proliferation, differentiation, apoptosis, and aging. In addition, ceramide, together with cholesterol and free fatty acids, constitutes intercellular lipids and plays an important role in the formation of the epidermal stratum corneum. However, ceramide is oil-soluble and recrystallizable, which limits its application and development in the fields of food, medicine, and cosmetics.

[0004] The accumulation of DNA telomere wear will lead to the increase of senescent cells in tissues and organs, resulting in the degradation of cell functions, which in turn will cause the body to decay and degenerate, and even the appearance of aging-related diseases. As the outermost tissue, the skin is the most intuitive external manifestation of body aging. Telomerase is an enzyme that synthesizes telomere DNA. Regulating telomerase activity is considered to be the key to anti-aging. Activating telomerase can slow down or even reverse the shortening of telomeres in cells, which helps cells divide and proliferate and prolongs life.

[0005] At present, most of the anti-aging active ingredients on the market are generally polyphenols, vitamins or peptides, which are sensitive to enzymes, light, heat, pH, etc., and are easily inactivated by environmental changes. In addition, the transdermal absorption effect of anti-aging active ingredients is difficult to guarantee, because the stratum corneum of the skin has a strong keratinocyte barrier function, which limits the penetration of external substances. Many active molecules, such as active peptides, are difficult to penetrate in the stratum corneum, thus affecting their skin care effect at the target site, and the anti-aging effect is minimal. Summary of the invention

[0006] The present invention provides a nanoliposome with the functions of resisting cell oxidation and inhibiting the activity of beta-galactosidase, and a preparation method and application thereof, so as to solve the problems of unstable action and unsatisfactory effect of anti-aging active ingredients in the prior art.

[0007] In a first aspect, the present invention provides a nanoliposome, comprising the following components: 8-12 wt % of cycloastragenol, 8-12 wt % of ceramide, 15-25 wt % of lecithin, 1-5 wt % of a proppant, 10-20 wt % of a solubilizer, and 10-25 wt % of a stabilizer.

[0008] As a possible implementation, the particle size of the nanoliposome is 100-200 nm.

[0009] As a possible implementation manner, the particle size is maintained at a temperature of -20 to 50° C. for at least 180 days.

[0010] In a second aspect, the present invention provides a method for preparing the nanoliposomes described in any possible implementation of the first aspect, comprising the following steps: uniformly dispersing the cycloastragenol, the ceramide, the lecithin, the proppant and the solubilizing agent in the oil phase at a temperature of 40 to 60° C., and keeping the temperature to obtain an oil phase dispersion system; uniformly dissolving the stabilizer in pure water at a temperature of 50 to 70° C., and keeping the temperature to obtain an aqueous phase dispersion system; under stirring conditions, dropwise adding the aqueous phase dispersion system to the oil phase dispersion system, shearing, and obtaining liposome colostrum; and subjecting the liposome colostrum to high-pressure homogenization to obtain the nanoliposomes.

[0011] As a possible implementation, the lecithin is a combination of one or more of hydrogenated lecithin, soybean lecithin, egg yolk lecithin and sunflower lecithin; and / or, the proppant is a combination of one or more of cholesterol, stigmasterol and β-sitosterol; and / or, the solubilizer is a combination of one or more of Tween 80, PEG-40 hydrogenated castor oil, polyethylene glycol 4000, polyethylene glycol fatty acid glycerate, polyglycerol-10 stearate, polysorbate and PEG / PPG-dimethyl ether; and / or, the stabilizer is a combination of one or more of glycerol, sorbitol, 1,3-propylene glycol, 1,3-butylene glycol, 1,2-pentanediol, 1,2-hexanediol and dipropylene glycol.

[0012] Exemplarily, the lecithin is hydrogenated lecithin; the proppant is cholesterol; the solubilizer is Tween 80 or PEG-40 hydrogenated castor oil; and the stabilizer is glycerol or sorbitol.

[0013] As a possible implementation method, the uniform dispersion in the oil phase is achieved by ultrasonic treatment for 5 to 10 minutes; and / or, the uniform dissolution in pure water is achieved by stirring at a rate of 200 to 400 rpm for 10 to 30 minutes; and / or, under stirring conditions, the rate is 300 to 500 rpm; and / or, the aqueous phase dispersion is added dropwise to the oil phase dispersion at a rate of 3 mL / min; and / or, the shearing conditions are 5000 to 10000 rpm, the duration is 5 to 15 minutes, and the temperature is 30 to 50°C; and / or, the high-pressure homogenization treatment is performed at a pressure of 80 to 100 MPa and the number of treatments is 3 to 6 times.

[0014] In a third aspect, the present invention provides a use of the nanoliposome described in any possible implementation of the first aspect or the nanoliposome prepared by the preparation method described in any possible implementation of the second aspect in the preparation of an aging inhibitor.

[0015] As a possible implementation, the nanoliposome is used to inhibit cell oxidative damage; and / or, the nanoliposome is used to inhibit cell inflammation; and / or, the nanoliposome is used to inhibit elastase.

[0016] In a fourth aspect, the present invention provides an aging inhibitor, comprising the nanoliposomes described in any possible implementation of the first aspect or the nanoliposomes prepared by the preparation method described in any possible implementation of the second aspect, and pharmaceutically acceptable excipients. Exemplarily, the excipients are solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, and release retardants, or a combination of several thereof.

[0017] As a possible implementation, the aging inhibitor is prepared into the following dosage forms: solution, suspension, emulsion, gas dispersion, microparticle dispersion or solid dispersion. Exemplarily, the aging inhibitor is prepared into an emulsion or a gel.

[0018] Cycloastragenol activates telomerase through the mitogen-activated protein kinase (MAPK) pathway, which activates the epidermal growth factor receptor (EGFR) by inducing phosphorylation of tyrosine 845 of c-Src protein, and then activates the Ras / MEK / ERK signaling pathway; cycloastragenol activates Ets transcription factor by inducing extracellular signal-regulated kinase (ERK) phosphorylation, thereby upregulating the expression of human telomerase reverse transcriptase gene (hTERT gene), thereby activating telomerase. Ceramide is a central molecule in sphingolipid metabolism, and the p38 mitogen-activated protein kinase (P38 MAPK) signal transduction pathway is the downstream pathway of ceramide; as a member of the MAPK family, p38 MAPK is an important signaling molecule with pro-apoptotic effects, and various environmental stresses and inflammatory cytokines can activate it; for other MAPK cascades, P38 MAPK is a MAPKKK, that is, a mixed lineage kinase (MLK); P38 MAPK phosphorylates and activates MKK3 / 6, which is an ERK kinase. Therefore, ceramide can induce ERK phosphorylation by activating the p38 MAPK signaling pathway, thereby enhancing the effect of cycloastragenol in activating telomerase.

[0019] The present invention provides a nanoliposome with anti-cell oxidation and inhibitory effects on the activity of beta-galactosidase, combined with the mechanism and action site of aging, ceramide and cycloastragalol are compounded, the activity of telomerase is significantly activated, the length of telomeres is extended, the tissue repair and regeneration process is enhanced, and aging is delayed. Wherein, ceramide, as a synergist of cycloastragalol, promotes the activation process of cycloastragalol to telomerase by activating the P38 MAPK signaling pathway. Ceramide and cycloastragalol have a synergistic effect, can strongly activate telomerase therapeutic targets through the MAPK pathway, extend the chromosome telomere region, maintain the integrity and stability of chromosomes, and play an important role in anti-aging and treating telomerase-dependent diseases. In addition, cycloastragalol can block the activation of inflammasomes through adenylate-activated protein kinase (AMPK), and ceramide regulates the activity of inflammation-related transcription factors and protein kinases through P38MAPK. The two jointly remove inflammatory factors, chemokines and growth factors secreted during aging through the MAPK pathway, and inhibit chronic inflammation associated with aging. They have complementary mechanisms and can effectively inhibit the inflammatory process and prevent oxidative damage and DNA damage caused by inflammation.

[0020] When amphiphilic lipid molecules such as phospholipids and sphingolipids are dispersed in the aqueous phase, the hydrophobic tails of the molecules tend to gather together, while the hydrophilic heads are exposed to the aqueous phase, and the spherical closed vesicles containing a bilayer structure are assembled to form, which are called nanoliposomes. This structure is similar to the human cell membrane. Therefore, through the encapsulation of nanoliposomes, drug molecules can quickly penetrate the skin barrier to reach the dermis and basal layer. In addition, the nanoliposome carrier can be fused into the cell membrane to accurately transport the drug to the targeted cells, and slowly release it, which not only improves the stability of the drug, but also prolongs the time for the drug to take effect. Ceramide and cycloastragenol are treated by nano-encapsulation, can be fused into the cell membrane, and can be accurately transported to the targeted cells, effectively solving the solubility and compatibility problems of cycloastragenol and ceramide. Lipid carriers can also control the sustained release of anti-aging active ingredients, promote the improvement of skin permeability and encapsulation rate, and achieve synergistic enhancement of anti-aging active ingredients. The substance finally shows good targeted activation of telomerase and inhibition of inflammatory response. The present invention uses nanoliposomes to encapsulate two functional ingredients, and the carrier has strong stability, good bioavailability, and is convenient to use.

[0021] The present invention provides a method for preparing nanoliposomes with the functions of resisting cell oxidation and inhibiting the activity of β-galactosidase. The method uses ultrasonic dispersion technology to disperse the active ingredients ceramide and cycloastragenol to improve the dispersibility of the active ingredients in the medium. Ultrasonic dispersion uses the cavitation effect generated by ultrasound to achieve the dispersion of solid particles in liquid. The cavitation effect can produce a high temperature and high pressure environment, which is conducive to the formation of nanoparticles. Ultrasonic dispersion can also reduce the binding force between particles and effectively prevent the occurrence of agglomeration. Mixing homogenization shearing can reduce the particle size of the dispersed phase in the dispersion system, obtain finer dispersed particles, and improve the uniformity and stability of the dispersion system. Ultrahigh pressure homogenization treatment can effectively make the dispersed phase more evenly dispersed in the dispersion system, and the homogenized product has better stability because the reduction of particle size can reduce the aggregation and precipitation between particles. For emulsified products, high pressure homogenization can improve the emulsification efficiency and make the oil-water mixture more stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 Schematic diagram of the cell antioxidant experiment results provided in an embodiment of the present invention; wherein, compared with the normal group, the H2O2 group ####P<0.0001; compared with the H2O2 group, *P<0.05, **P<0.01, ***P<0.005, ****P<0.0001.

[0024] Figure 2 Schematic diagram of the cell anti-inflammatory experiment results provided in an embodiment of the present invention; wherein, the LPS group compared with the normal group ####P<0.0001; compared with the LPS group *P<0.05, **P<0.01, ***P<0.005, ****P<0.0001.

[0025] Figure 3 A schematic diagram of the zebrafish anti-aging experimental results provided by an embodiment of the present invention.

[0026] Figure 4 Schematic diagram of the experimental results of elastase inhibition rate provided in an embodiment of the present invention; wherein, the negative control is compared with the positive control ####P<0.0001; compared with the negative control *P<0.05, **P<0.01, ***P<0.005, ****P<0.0001.

[0027] Figure 5A schematic diagram of the chicken embryo chorioallantoic membrane experimental results provided in an embodiment of the present invention, wherein, from top to bottom, there are a blank control group, a negative control group, Example 1 group, Example 2 group, and Example 3 group, with 0 min on the left and 5 min on the right. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not 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 are within the scope of protection of the present invention.

[0029] In order to solve the problem that the anti-aging active ingredients in the prior art have unstable effects and unsatisfactory effects, the present invention provides a nanoliposome with the effects of resisting cell oxidation and inhibiting the activity of β-galactosidase.

[0030] Cycloastragenol activates telomerase through the mitogen-activated protein kinase (MAPK) pathway, which activates the epidermal growth factor receptor (EGFR) by inducing phosphorylation of tyrosine 845 of c-Src protein, and then activates the Ras / MEK / ERK signaling pathway; cycloastragenol activates Ets transcription factor by inducing extracellular signal-regulated kinase (ERK) phosphorylation, thereby upregulating the expression of human telomerase reverse transcriptase gene (hTERT gene), thereby activating telomerase. Ceramide is a central molecule in sphingolipid metabolism, and the p38 mitogen-activated protein kinase (P38 MAPK) signal transduction pathway is the downstream pathway of ceramide; as a member of the MAPK family, p38 MAPK is an important signaling molecule with pro-apoptotic effects, and various environmental stresses and inflammatory cytokines can activate it; for other MAPK cascades, P38 MAPK is a MAPKKK, that is, a mixed lineage kinase (MLK); P38 MAPK phosphorylates and activates MKK3 / 6, which is an ERK kinase. Therefore, ceramide can induce ERK phosphorylation by activating the p38 MAPK signaling pathway, thereby enhancing the effect of cycloastragenol in activating telomerase.

[0031] Furthermore, the embodiment of the present invention provides a stability test experiment of a nanoliposome with anti-cellular oxidation and β-galactosidase activity inhibition effect, which verifies that the particle size is uniform, the size is moderate, the amplitude of change over time is smaller, and the system is more stable.

[0032] Furthermore, the embodiments of the present invention provide a test experiment for the cellular antioxidant capacity, anti-inflammatory capacity, anti-aging capacity and elastase inhibition rate of a nanoliposome with anti-cellular oxidation and β-galactosidase activity inhibition effects, which verifies that it has strong antioxidant damage performance, strong anti-inflammatory capacity, strong anti-aging capacity and higher elastase inhibition rate.

[0033] Furthermore, the embodiment of the present invention also provides a test experiment of the irritation of the nanoliposomes with anti-cellular oxidation and β-galactosidase activity inhibition effects, which verifies that the nanoliposomes are non-irritating.

[0034] The technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0035] Example 1

[0036] This example provides an experiment for preparing nanoliposomes with the effects of resisting cell oxidation and inhibiting β-galactosidase activity.

[0037] 12.0g of cycloastragenol, 10.0g of ceramide, 20.0g of hydrogenated lecithin, 3.0g of cholesterol and 15.0g of Tween 80 were ultrasonically treated at 55°C for 8min to obtain a uniform oil phase, and the mixture was kept warm for 15min. 20.0g of glycerol was dissolved in pure water, the temperature was raised to 60°C, and magnetically stirred at 300rpm for 20min to obtain a uniform water phase, and the mixture was kept warm for 15min. Under stirring at 400rpm, the uniform water phase was added dropwise to the uniform oil phase at a speed of 3mL / min, and the mixture was sheared at 8000rpm for 10min, with the shear temperature at 40°C, and the mixture was kept warm for 15min to obtain liposome colostrum. The liposome colostrum was subjected to high-pressure homogenization treatment at a pressure of 100Mpa for 4 times to obtain nanoliposomes.

[0038] Example 2

[0039] This example provides an experiment for preparing nanoliposomes with the effects of resisting cell oxidation and inhibiting β-galactosidase activity.

[0040] 12.0g of cycloastragenol, 10.0g of ceramide, 15.0g of soybean lecithin, 2.0g of stigmasterol and 20.0g of PEG-40 hydrogenated castor oil were ultrasonically treated at 60°C for 5min to obtain a uniform oil phase, and the mixture was kept warm for 15min; 15.0g of sorbitol was dissolved in pure water, the temperature was raised to 70°C, and magnetically stirred at 200rpm for 15min to obtain a uniform aqueous phase, and the mixture was kept warm for 15min; under stirring at 500rpm, the uniform aqueous phase was added dropwise to the uniform oil phase at a speed of 3mL / min, and high-speed shearing was performed at 10000rpm for 5min, the shear temperature was 45°C, and the mixture was kept warm for 15min to obtain liposome colostrum; the liposome colostrum was high-pressure homogenized at a pressure of 80Mpa for 6 times to obtain nanoliposomes.

[0041] Example 3

[0042] This example provides an experiment for preparing nanoliposomes with the effects of resisting cell oxidation and inhibiting β-galactosidase activity.

[0043] 12.0g of cycloastragenol, 10.0g of ceramide, 25.0g of egg yolk lecithin, 5.0g of β-sitosterol and 10.0g of PEG-40 polyethylene glycol 4000 were ultrasonically treated for 10 minutes at 45°C to obtain a uniform oil phase, and the mixture was kept warm for 15 minutes; 25.0g of 1,3-propylene glycol was dissolved in pure water, the temperature was raised to 50°C, and magnetically stirred at 400rpm for 25 minutes to obtain a uniform aqueous phase, and the mixture was kept warm for 15 minutes; under stirring at 300rpm, the uniform aqueous phase was added dropwise to the uniform oil phase at a rate of 3mL / min, and high-speed shearing was performed at 5000rpm for 15 minutes, the shear temperature was 35°C, and the mixture was kept warm for 15 minutes to obtain liposome colostrum; the liposome colostrum was high-pressure homogenized for 5 times at a pressure of 90Mpa to obtain nanoamine liposomes.

[0044] Comparative Example 1

[0045] This example provides an experiment for preparing liposomes containing cycloastragenol.

[0046] 22.0g of cycloastragenol, 20.0g of hydrogenated lecithin, 3.0g of cholesterol and 15.0g of Tween 80 were ultrasonically treated at 55°C for 8min to obtain a uniform oil phase, and the mixture was kept warm for 15min; 20.0g of glycerol was dissolved in pure water, the temperature was raised to 60°C, and magnetically stirred at 300rpm for 20min to obtain a uniform water phase, and the mixture was kept warm for 15min; under stirring at 400rpm, the uniform water phase was added dropwise to the uniform oil phase at a speed of 3mL / min, and high-speed shearing was performed at 8000rpm for 10min, the shear temperature was 40°C, and the mixture was kept warm for 15min to obtain liposome colostrum; the liposome colostrum was subjected to high-pressure homogenization treatment at a pressure of 100Mpa for 4 times to obtain liposomes containing cycloastragenol.

[0047] Comparative Example 2

[0048] This example provides an experiment for preparing ceramide liposomes.

[0049] 22.0g ceramide, 20.0g hydrogenated lecithin, 3.0g cholesterol and 15.0g Tween 80 were ultrasonically treated at 55°C for 8min to obtain a uniform oil phase, and the mixture was kept warm for 15min; 20.0g glycerol was dissolved in pure water, the temperature was raised to 60°C, and magnetically stirred at 300rpm for 20min to obtain a uniform aqueous phase, and the mixture was kept warm for 15min; under stirring at 400rpm, the uniform aqueous phase was added dropwise to the uniform oil phase at a rate of 3mL / min, and high-speed shearing was performed at 8000rpm for 10min, the shear temperature was 40°C, and the mixture was kept warm for 15min to obtain liposome colostrum; the liposome colostrum was subjected to high-pressure homogenization at a pressure of 100Mpa for 4 times to obtain ceramide liposomes.

[0050] Comparative Example 3

[0051] This example provides an experiment for preparing a mixture of cycloastragenol and ceramide.

[0052] 12.0 g of cycloastragenol and 10.0 g of ceramide were added to 78 g of pure water, and stirred until the mixture was uniform, thereby obtaining a mixture of cycloastragenol and ceramide.

[0053] Test Example 1

[0054] This test example provides the appearance and particle size stability test experiments of each product.

[0055] Stability is the ability to maintain its initial properties or properties without changing over time under specified or reasonably foreseeable storage and use conditions. The test process for appearance and particle size stability is to place the test object in a sealed container, place it at a specific temperature for a specific period of time, and check the appearance and particle size of the test object at a specific time point.

[0056] The products prepared in Examples 1 to 3 and Comparative Examples 1 to 3 were respectively used as test objects, and were placed at -18°C, 4°C, room temperature, and 45°C for 180 days. The appearance of the samples was checked at room temperature for precipitation or discoloration in the initial state, 90 days, and 180 days later, and the particle size of the samples was tested. The results shown in Table 1 were obtained.

[0057] Table 1 Appearance and particle size stability test results of each product

[0058]

[0059] As can be seen from Table 1, the products prepared in Examples 1 to 3 and Comparative Examples 1 to 2 were stored at different temperatures (45°C, room temperature, 4°C, -20°C) for 180 days without agglomeration or stratification; and the product prepared in Example 1 had a smaller particle size than the products of Examples 2, 3, Comparative Examples 1 and 2, and the amplitude of the change over time was smaller, and the system was more stable. It can be seen that the nanoliposomes provided by the present invention have a good particle size range and stability.

[0060] Test Example 2

[0061] This test example provides a test experiment for the cellular antioxidant capacity of each product.

[0062] HaCaT cells in logarithmic growth phase were cultured at 8×10 3 / well was inoculated in a 96-well plate, with a system of 100 μL per well. The outermost circle of the 96-well plate was sealed with 100 μL of PBS. After the cells were completely attached to the wall, the matrix to be tested was replaced. After the cells were cultured for 24 hours, the cell activity was detected by CCK-8 method. After that, hydrogen peroxide dilution (diluted to 800 μM with DMEM medium) was given, and the model was cultured in an incubator for 4 hours. After 4 hours, the cell activity was immediately detected by CCK8 method.

[0063] The products prepared in Examples 1 to 3 and Comparative Examples 1 to 3 were diluted with DMEM medium to a final concentration of 10 μg / mL of active ingredients, and the matrix to be tested was added; a blank group without hydrogen peroxide damage and a H2O2 group without drug administration were also set up. The cellular antioxidant capacity of each product was tested, and the results were as follows: Figure 1 Results shown.

[0064] Depend on Figure 1It can be seen that after hydrogen peroxide damage, the cell viability of the H2O2 group was 41.3%, indicating that the hydrogen peroxide damage HaCaT model was successfully established. Compared with the blank group, the products prepared in Examples 1 to 3 and Comparative Examples 1 to 2 can significantly improve the viability of oxidatively damaged HaCaT cells, and the difference is statistically significant (P < 0.01). Among them, the product prepared in Example 1 has the strongest antioxidant damage performance, and the effects of Examples 1 to 3 are better than those of the comparative examples. It can be seen that Example 1 has the best antioxidant properties.

[0065] Test Example 3

[0066] This test example provides a test experiment for the cellular anti-inflammatory ability of each product.

[0067] RAW264.7 cells in the logarithmic growth phase were inoculated in a 24-well plate at a cell density of 2×10 5 ~5×10 5 / mL, 0.5mL per well, incubate for 24h. After 24h, remove the supernatant and add 0.5mL of the following samples respectively: ① Normal group: DMEM medium (2% FBS; 1% double antibody); ② LPS group: 1μg / mL LPS; ③ Experimental group: test substance (1%) + LPS (1μg / mL). After acting on the cells for 24h, use a pipette to aspirate the supernatant into a centrifuge tube, centrifuge at 1000r / min for 5min, discard the precipitate and aspirate the supernatant, and use TNF-α and IL-6 inflammatory factor detection kits to detect the changes in the amount of inflammatory factors in the supernatant. The detection method strictly follows the instructions on the detection kit.

[0068] The products prepared in Examples 1 to 3 and Comparative Examples 1 to 3 were used as test objects to detect the anti-inflammatory ability of cells. Figure 2 Results shown.

[0069] Depend on Figure 2It can be seen that the levels of TNF-α and IL-6 in the LPS group were significantly higher than those in the normal group, indicating that the inflammatory induction model was successfully established. Examples 1 to 3 and Comparative Examples 1 to 3 can all cause a significant decrease in the level of inflammatory factor TNF-α in RAW 264.7 cells, and the difference is statistically significant (P < 0.01), and the effects of Example 1, Example 2, and Example 3 in inhibiting the inflammatory factor TNF-α are better than those in the comparative example; Examples 1 to 3, Comparative Examples 1 and 2 can all cause a significant decrease in the level of inflammatory factor IL-6 in RAW264.7 cells, and the difference is statistically significant (P < 0.01), and the effects of Example 1, Example 2, and Example 3 in inhibiting the inflammatory factor IL-6 are better than those in the comparative example. It can be seen that the products prepared by Examples 1 to 3 can significantly reduce the content of inflammatory factors in cells, and the effects are stronger than those of Comparative Examples 1 to 3; among them, the product prepared by Example 1 has the strongest effect in inhibiting the expression of inflammatory factors.

[0070] Test Example 4

[0071] This test example provides a test experiment for the anti-aging ability of each product.

[0072] Current studies have shown that the causes of skin aging include: accumulation of reactive oxygen species, loss of collagen and elastin, increased activity of β-galactosidase, etc. Therefore, the β-galactosidase staining method can detect the degree of cell aging, and the lower the staining intensity, the lower the degree of aging, and the stronger the anti-aging effect of the test object. The specific steps of the β-galactosidase staining method are: select 6hpf zebrafish with the same developmental stage, place them in a covered culture dish with standard dilution water, add a certain concentration of the test sample, perform β-galactosidase staining after 120 hours, and take pictures after incubation overnight. The normal control group is standard dilution water, and the model group is 300μmol / L hydrogen peroxide.

[0073] The products prepared in Examples 1 to 3 were used as test samples to test their anti-aging abilities. Figure 3 Results shown.

[0074] Depend on Figure 3 It can be seen that the staining intensity of Example 1, Example 2, and Example 3 is significantly lower than that of the model group, and is close to that of the normal control group; among them, the staining intensity of Example 1 is closest to that of the normal control group. It can be seen that the products prepared in Example 1, Example 2, and Example 3 all have anti-aging properties, and the product prepared in Example 1 has the strongest anti-aging effect.

[0075] Test Example 5

[0076] This test example provides a test experiment for the elastase inhibition rate of each product.

[0077] Add 1000 μL buffer, 50 μL test sample diluent and 50 μL elastase solution to a 96-well plate, mix well and incubate at 25°C for 20 minutes. Then add 50 μL substrate and incubate for 60 minutes to detect the absorbance, calculate the effect of the test sample on the activity of elastase and calculate the statistical difference P value.

[0078] The products prepared in Examples 1 to 3 were used as test samples, respectively, and diluted with pure water to a concentration of 10%, and treated with 50 μmol / L sivelestat sodium (tetrahydrate) as a positive control group, and treated with pure water as a negative control group. Three parallel experiments were set up for each treatment to test the elastase inhibition rate. The results are as follows: Figure 4 And the results shown in Table 2.

[0079] Table 2 Elastase inhibition rate (%)

[0080] Repeat 1 Repeat 2 Repeat 3 Mean p-value Negative control group 0.91 1.13 0.72 0.92 / Example 1 51.31 50.41 52.39 51.37 p<0.05 Example 2 50.49 49.27 51.18 50.31 p<0.05 Example 3 50.10 49.13 48.39 49.21 p<0.05 Comparative Example 1 5.39 5.19 5.04 5.21 p<0.05 Comparative Example 2 4.31 3.32 4.98 4.20 p<0.05 Comparative Example 3 3.21 4.30 3.19 3.57 p<0.05 Positive control group 78.62 79.31 76.42 78.12 p<0.05

[0081] Depend on Figure 4 As shown in Table 2, compared with the negative control group, the elastase inhibition rate of the products prepared in Examples 1 to 3 is higher, and the elastase inhibition rate of the product prepared in Example 1 is the highest. It can be seen that the products prepared in Examples 1 to 3 have the effect of firming and anti-wrinkle.

[0082] Test Example 6

[0083] This test example provides irritation test experiments of various products.

[0084] After removing the eggshell and inner membrane of the air chamber of SPF eggs incubated for 9 to 12 days, use a pipette to slowly drip 0.3 mL of the sample to be tested into the chorioallantoic membrane (CAM). The blank control group was dripped with 0.3 mL of 0.9% saline, and the positive control group was dripped with 0.3 mL of 0.1 mol / L sodium hydroxide. The reaction changes of CAM were observed within 5 minutes and recorded by taking photos.

[0085] The products prepared in Examples 1 to 3 were used as samples to be tested, and their irritation was tested. Figure 5 Results shown.

[0086] Depend on Figure 5 It can be seen that the products prepared in Examples 1 to 3 had no bleeding, no coagulation, and no vascular lysis after acting on CAM for 5 minutes. It can be seen that the products prepared in Examples 1 to 3 had no irritation.

[0087] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0088] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A nanoliposome, characterized in that: The composition includes: 8-12wt% cycloastragenol, 8-12wt% ceramide, 15-25wt% lecithin, 1-5wt% proppant, 10-20wt% solubilizer, and 10-25wt% stabilizer; The lecithin is one of hydrogenated lecithin, soybean lecithin and egg yolk lecithin; The proppant is one of cholesterol, stigmasterol and β-sitosterol; The solubilizing agent is one of Tween 80, PEG-40 hydrogenated castor oil and polyethylene glycol 4000; The stabilizer is one of glycerol, sorbitol and 1,3-propylene glycol.

2. The nanoliposome according to claim 1, characterized in that Its particle size is 100~200nm.

3. The nanoliposome according to claim 2, characterized in that The particle size is maintained at a temperature of -20 to 50°C for at least 180 days.

4. A method for preparing nanoliposomes according to any one of claims 1 to 3, characterized in that: The following steps are involved: The cycloastragenol, the ceramide, the lecithin, the proppant and the solubilizing agent are uniformly dispersed in the oil phase at a temperature of 40 to 60° C., and the temperature is kept to obtain an oil phase dispersion system; The stabilizer is uniformly dissolved in pure water at a temperature of 50-70° C., and the temperature is kept to obtain an aqueous phase dispersion system; Under stirring conditions, adding the aqueous phase dispersion system dropwise to the oil phase dispersion system, and shearing, to obtain liposome colostrum; The liposome colostrum is subjected to high pressure homogenization to obtain the nanoliposome.

5. The preparation method according to claim 4, characterized in that: The uniform dispersion into the oil phase is achieved by ultrasonic treatment for 5 to 10 minutes; And / or, the uniform dissolution in pure water is achieved by stirring at a rate of 200-400 rpm for 10-30 min; And / or, under the stirring condition, the speed is 300-500 rpm; and / or, adding the aqueous phase dispersion system dropwise to the oil phase dispersion system at a rate of 3 mL / min; And / or, the shearing conditions are 5000-10000 rpm, 5-15 min, and 30-50° C.; And / or, the high pressure homogenization treatment has a pressure of 80-100 MPa and is performed 3-6 times.

6. Use of the nanoliposome according to any one of claims 1 to 3 or the nanoliposome prepared by the preparation method according to any one of claims 4 to 5 in the preparation of an aging inhibitor.

7. The use according to claim 6, characterized in that: The nanoliposomes are used to inhibit cellular oxidative damage; and / or, the nanoliposomes are used to inhibit cellular inflammation; And / or, the nanoliposomes are used to inhibit elastase.

8. An aging inhibitor, characterized in that The invention comprises the nanoliposome according to any one of claims 1 to 3 or the nanoliposome prepared by the preparation method according to any one of claims 4 to 5, and pharmaceutically acceptable excipients.

9. The aging inhibitor according to claim 8, characterized in that Prepared into the following dosage form: Solutions, suspensions, emulsions, gas dispersions, microparticle dispersions or solid dispersions.

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