A skin anti-aging repair composition and multi-layer lipid nanoparticles containing the same

Abstract

The present invention relates to the field of cosmetics, and the present invention discloses a skin anti-aging repair composition and a multilayer lipid nanoparticle comprising the same. First, the composition provided by the present invention comprises palmitoyl tripeptide-8 and Rosa centifolia flower extract, which has a synergistic effect in skin anti-aging repair; secondly, the multilayer lipid nanoparticle provided by the present invention loads palmitoyl tripeptide-8 and Rosa centifolia flower extract in different layers, and successfully achieves isolation of the two; in addition, the embedding of palmitoyl tripeptide-8 and Rosa centifolia flower extract by the multilayer lipid nanoparticle can not only significantly improve the storage stability of active substances, but also greatly improve the transdermal properties of active substances.

Description

Technical Field

[0001] The invention relates to the field of cosmetics, and in particular to a skin anti-aging and repairing composition and multi-layer lipid nanoparticles containing the same. Background Art

[0002] The human skin is rich in nerves, and there is a close connection between nerve fibers, keratinocytes and melanocytes. Neuropeptides secreted by nerve cells, skin cells and immune cells act as messengers to maintain the dynamic balance of the skin. When this balance is broken by internal or external invasion, it will cause a series of skin problems such as light lines, lack of skin elasticity and dull complexion. Palmitoyl tripeptide-8 is a neurotransmitter inhibitory peptide that can inhibit the release of Substance P and pro-inflammatory cytokines, thereby restoring the dynamic balance of the skin and improving skin conditions.

[0003] Rosa centifolia flower extract is mainly used as a skin conditioner and antioxidant in the field of cosmetics and skin care products. Rosa centifolia flower extract is rich in compounds, including organic acids, vitamins, sugars, flavonoids, alkaloids, minerals, proteins, amino acids, fats, etc., among which organic acid compounds are its main active ingredients. Studies have shown that Rosa centifolia flower extract can promote the production of ceramides, thereby repairing and softening the skin; it can quench free radicals and improve dull and aging skin.

[0004] The factors that lead to skin aging are multifaceted and have a certain degree of complexity, so skin anti-aging also requires the synergistic effect of multiple active substances with different mechanisms of action. In the art, compounding active substances with different mechanisms of action may produce unexpected effects. In the prior art, there is no shortage of reports and products that add palmitoyl tripeptide-8 or Rosa centifolia flower extract to cosmetics and skin care products to exert skin anti-aging effects. However, there are currently no related reports or products that use the above two in combination. Therefore, it is of certain significance to study whether palmitoyl tripeptide-8 and Rosa centifolia flower extract have a synergistic effect on skin anti-aging.

[0005] In the preliminary research of the present invention, it was found that the compounding of palmitoyl tripeptide-8 and Rosa centifolia flower extract has the following problems to be solved:

[0006] (1) Palmitoyl tripeptide-8 has multiple amine groups and is an alkaline substance; while the main active ingredient in Rosa centifolia flower extract, the organic acid compound, contains carboxyl groups and is an acidic substance. If the two are directly combined, neutralization will easily occur, thus affecting the anti-aging effect. Therefore, how to integrate palmitoyl tripeptide-8 and Rosa centifolia flower extract, which have different mechanisms of action and physicochemical properties, into the same preparation and deliver them together to the skin anti-aging target tissues or target cells is a difficult problem.

[0007] (2) Rosa centifolia flower extract contains a variety of active ingredients, among which organic acids are the main anti-aging and antioxidant ingredients. Organic acids are hydrophilic and easily blocked by the skin barrier, making it difficult for them to penetrate deep into the skin to take effect. Palmitoyl tripeptide-8 is an oligopeptide composed of three amino acids and has a lipophilic palmitoyl group, which makes it easier for it to penetrate the skin than organic acid compounds.

[0008] (3) Palmitoyl tripeptide-8 has poor stability and is easily degraded and loses its activity during storage due to environmental factors. Summary of the invention

[0009] In order to solve the above technical problems, the present invention provides a skin anti-aging repair composition and a multilayer lipid nanoparticle containing the same. First, the composition provided by the present invention contains a specific ratio of palmitoyl tripeptide-8 and Rosa centifolia flower extract, which has a synergistic effect in skin anti-aging repair; secondly, the multilayer lipid nanoparticles provided by the present invention load palmitoyl tripeptide-8 and Rosa centifolia flower extract in different layers, successfully achieving isolation of the two; in addition, the multilayer lipid nanoparticles embedding palmitoyl tripeptide-8 and Rosa centifolia flower extract can not only significantly improve the storage stability of active substances (especially palmitoyl tripeptide-8), but also greatly improve the transdermal properties of active substances.

[0010] The specific technical scheme of the present invention is:

[0011] In a first aspect, the present invention provides a skin anti-aging and repairing composition, comprising palmitoyl tripeptide-8 and Rosa centifolia flower extract in a mass ratio of 1:0.1-1:100. After separation and purification, the total content of organic acid compounds in the Rosa centifolia flower extract is at least 80wt%.

[0012] The present invention explores for the first time the effect of palmitoyl tripeptide-8 and rosa centifolia flower extract on skin anti-aging and repair after compounding, and the results show that the two have a synergistic effect. Among them, palmitoyl tripeptide-8 reduces the release of neurotransmitter substance P and pro-inflammatory cytokines; thereby reducing vasodilation, edema and erythema, improving skin sensitivity and maintaining skin health. As a beauty ingredient with a long history, rosa centifolia flower extract has always been considered to have the effects of fragrance, brightening and nourishing. However, it was found that after combining the two, they can not only play their original anti-inflammatory, soothing, nourishing and brightening effects, but also have higher activity and efficacy in anti-wrinkle and firming.

[0013] Preferably, the mass ratio of the palmitoyl tripeptide-8 to the Rosa centifolia flower extract is further preferably 1:1-1:10.

[0014] In a second aspect, the present invention provides a cosmetic or skin care product containing the above composition and having skin anti-aging and repairing effects.

[0015] Preferably, the cosmetics or skin care products are skin care products such as face cream, lotion, scrub, massage cream, essence, makeup remover, cleanser, and color makeup products such as liquid foundation, pressed powder, lipstick, eye shadow, etc.

[0016] In a third aspect, the present invention provides a multi-layer lipid nanoparticle comprising palmitoyl tripeptide-8 and Rosa centifolia flower extract, comprising:

[0017] An aqueous core containing Rosa centifolia flower extract, a double lipid middle layer containing palmitoyl tripeptide-8 and nanopores coated on the surface of the aqueous core by electrostatic action, an aqueous middle layer containing Rosa centifolia flower extract coated on the surface of the double lipid middle layer by electrostatic action, and an outer lipid layer containing palmitoyl tripeptide-8 coated on the surface of the aqueous middle layer by electrostatic action.

[0018] As described in the background technology section, there is no related report on integrating palmitoyl tripeptide-8 and Rosa centifolia flower extract into the same preparation. After preliminary exploration, the present invention found that there are three technical difficulties in preparing palmitoyl tripeptide-8 and Rosa centifolia flower extract into a preparation: ① Palmitoyl tripeptide-8 and Rosa centifolia flower extract have poor compatibility; ② The preparation is difficult to penetrate the skin stratum corneum barrier and has a poor transdermal effect (especially Rosa centifolia flower extract); ③ Poor stability and easy degradation and inactivation (especially palmitoyl tripeptide-8).

[0019] To this end, the present invention designs and prepares a multilayer lipid nanoparticle having a multilayer coating structure, wherein the core is an aqueous phase inner core containing a Rosa centifolia flower extract, the outer layer is a double lipid middle layer containing palmitoyl tripeptide-8 and nanopores, the outer layer is an aqueous phase middle layer containing a Rosa centifolia flower extract, and the outer layer is a lipid outer layer containing palmitoyl tripeptide-8.

[0020] In terms of isolation, in the above-mentioned multilayer lipid nanoparticle structure, the Rosa centifolia flower extract and palmitoyl tripeptide-8 are isolated in different layer structures, thereby avoiding direct contact between the two.

[0021] In terms of transdermal permeability, due to the large molecular weight and polarity, active substances (especially Rosa centifolia flower extract) generally have the defect of being difficult to penetrate the skin stratum corneum barrier and smoothly delivered to the skin anti-aging repair target tissue or target cell. For this reason, in the prior art, active substances are usually loaded in liposomes (aqueous phase inner core + double-layer lipid shell). Since the structure of the double-layer lipid shell is similar to the cell membrane structure, liposomes can significantly improve the skin permeability of active substances as nanocarriers. However, the present invention finds that the ability of liposomes as nanocarriers in penetrating the skin stratum corneum barrier still needs to be improved. The reason is that the skin barrier of the human body also has a multilayer structure, which is mainly hydrophobic and partially hydrophilic. The number of layers of the liposome structure is less, so its ability to penetrate the multilayer structure of the human skin barrier is limited. The number of layers of the multilayer lipid nanoparticles of the present invention is more than conventional liposomes. In the process of penetrating the multilayer structure of the human skin barrier, when encountering the hydrophobic layer of the skin barrier, the lipid outer layer of the multilayer lipid nanoparticles dissolves, and the palmitoyl tripeptide-8 therein is released; when continuing to encounter the hydrophilic layer of the skin barrier, the aqueous phase middle layer in the outer layer of the multilayer lipid nanoparticles disintegrates, and the aqueous Rosa centifolia flower extract is released; when continuing to encounter the hydrophobic layer and the hydrophilic layer of the skin barrier, according to the same similar compatibility principle, the double lipid middle layer and the aqueous phase inner core of the multilayer lipid nanoparticles disintegrate in sequence and release palmitoyl tripeptide-8 and Rosa centifolia flower extract respectively. In summary, the multilayer lipid nanoparticles of the present invention can penetrate through a thicker skin barrier.

[0022] In terms of stability, since the molecular structure of the Rosa centifolia flower extract carries a carboxyl group, it is positively charged at a specific acidic pH, and palmitoyl tripeptide-8 carries multiple amine groups, which is negatively charged at a specific alkaline pH. The present invention cleverly utilizes this material property, and can make the adjacent inner and outer layers combine through electrostatic adsorption during preparation, thereby increasing the enrichment rate and coating amount of the outer layer on the inner surface, and can also improve the structural stability of the multilayer lipid nanoparticles themselves, thereby achieving a better encapsulation effect on the active substance, making the stability of the active substance better. In addition, if the multilayer lipid nanoparticles of the present invention are prepared as a liquid suspension preparation, it is easy to settle and stratify after standing for a long time. For this reason, the bilayer lipid middle layer of the multilayer lipid nanoparticles of the present invention is designed with a nanopore structure, and these nanopores can significantly improve the suspension ability of the multilayer lipid nanoparticles, can reduce the sedimentation and stratification phenomenon, and improve storage stability.

[0023] In a fourth aspect, the present invention provides a method for preparing the multi-layer lipid nanoparticles, comprising the following steps:

[0024] (1) The Rosa centifolia flower extract is dissolved in water and glycerol, and the pH is adjusted to 5-6 to obtain a positively charged aqueous core liquid; palmitoyl tripeptide-8 is uniformly stirred with lecithin, cholesterol and ethanol aqueous solution at 40-45°C, and the pH is adjusted to 8.0-9.0 with sodium bicarbonate and sodium carbonate to obtain a negatively charged double-layer lipid intermediate layer liquid; the negatively charged double-layer lipid intermediate layer liquid is quickly injected into the aqueous core liquid, the ethanol is evaporated, ultrasound is performed, dialyzed, freeze-dried, and heated at 50-55°C to form nanopores, thereby obtaining lipid nanoparticles A having a double-layer lipid intermediate layer encapsulating an aqueous core structure; wherein the aqueous core is loaded with the Rosa centifolia flower extract, and the double-layer lipid intermediate layer is loaded with palmitoyl tripeptide-8.

[0025] (2) Dispersing lipid nanoparticles A in anhydrous ethanol and adjusting the pH to 7.5-8.0 to obtain a negatively charged suspension A; dissolving Rosa centifolia flower extract in water and glycerol and adjusting the pH to 3.5-4.5 to obtain a positively charged aqueous intermediate layer liquid; adding the suspension A dropwise to the aqueous intermediate layer liquid under stirring conditions, dialyzing, and freeze-drying to obtain lipid nanoparticles B whose outermost layer is an aqueous intermediate layer; wherein the aqueous intermediate layer is loaded with Rosa centifolia flower extract.

[0026] (3) Dispersing lipid nanoparticles B in water, adjusting the pH to 3.5-4.5, and obtaining a positively charged suspension B; stirring palmitoyl tripeptide-8, lecithin, cholesterol, and anhydrous ethanol at 40-45°C, and adjusting the pH to 7.5-8.0, to obtain a negatively charged lipid outer layer liquid; under stirring conditions, adding the suspension B dropwise to the lipid outer layer liquid, dialyzing, and freeze-drying to obtain multilayer lipid nanoparticles whose outermost layer is a lipid outer layer; wherein the lipid outer layer is loaded with palmitoyl tripeptide-8.

[0027] In order to allow each layer to be combined through electrostatic interaction during the preparation process, the pH of each solution or suspension needs to be strictly controlled. If the pH is too high or too low, it is not only easy to cause the Rosa centifolia flower extract and palmitoyl tripeptide-8 in the solution or suspension to be uncharged or oppositely charged, but it is also easy for the opposite charge to be excessively enriched on the surface due to the strong charge, resulting in the loss of control of the particle size of the nanoparticles. Specifically, in steps (2) and (3), it is necessary to control the pH of the aqueous phase intermediate layer liquid and suspension B to 3.5-4.5, and the pH of suspension A and lipid outer layer liquid to 7.5-8.0. Since step (1) requires the formation of nanopores in the bilayer lipid intermediate layer, the present invention selects sodium bicarbonate and sodium carbonate as pH regulators in step (1). Under heating conditions of 50-55°C, sodium bicarbonate is thermally decomposed to release gas to form nanopores in the bilayer lipid intermediate layer. It should be noted that in order to ensure that sodium bicarbonate is in excess, it is necessary to increase the pH of the bilayer lipid intermediate layer liquid in step (1) to 8.0-9.0, while lowering the pH of the aqueous core liquid to 5-6, and ensure that the bilayer lipid intermediate layer liquid is in excess during mixing.

[0028] By setting the above process parameters, the present invention cleverly utilizes the material specificity of the Rosa centifolia flower extract and palmitoyl tripeptide-8, and can make the adjacent inner and outer layers combine through electrostatic adsorption during preparation, thereby increasing the enrichment rate and coating amount of the outer layer on the inner surface, and at the same time, it can also improve the structural stability of the multilayer lipid nanoparticles themselves, thereby achieving a better encapsulation effect on the active substance, making the stability of the active substance better. In addition, the bilayer lipid middle layer of the multilayer lipid nanoparticles of the present invention is designed with a nanopore structure, and these nanopores can significantly improve the suspension ability of the multilayer lipid nanoparticles, reduce the sedimentation and stratification phenomenon, and improve storage stability.

[0029] Preferably, in step (1), in the aqueous core liquid, the mass ratio of Rosa centifolia flower extract, water and glycerin is 1:(85-95):(5-15).

[0030] Preferably, in step (1): in the bilayer lipid intermediate layer liquid, the mass ratio of palmitoyl tripeptide-8, lecithin, cholesterol and ethanol aqueous solution is 1: (5-10): (2-4): (100-200); and the ethanol concentration in the ethanol aqueous solution is 80-95wt%.

[0031] Preferably, in step (1), the mass ratio of the inner core liquid in the aqueous phase to the bilayer lipid middle layer liquid is 1:(3-5).

[0032] Preferably, in step (2): in the suspension A, the mass ratio of lipid nanoparticles A to anhydrous ethanol is 1:(50-150).

[0033] Preferably, in step (2), in the aqueous intermediate layer liquid, the mass ratio of Rosa centifolia flower extract, water and glycerin is 1:(85-95):(5-15).

[0034] Preferably, in step (2), the mass ratio of the suspension A to the aqueous intermediate layer liquid is 1:(2-3).

[0035] Preferably, in step (3): in the suspension B, the mass ratio of lipid nanoparticles B to water is 1:(50-150).

[0036] Preferably, in step (3): in the lipid outer layer liquid, the mass ratio of palmitoyl tripeptide-8, lecithin, cholesterol and anhydrous ethanol is 1:(5-10):(2-4):(100-200).

[0037] Preferably, in step (3), the mass ratio of the suspension B to the lipid outer layer liquid is 1:(2-4).

[0038] Preferably, the particle size of the multilayer lipid nanoparticles is 50-100 nm.

[0039] Compared with the prior art, the present invention has the following technical effects:

[0040] (1) The composition provided by the present invention contains palmitoyl tripeptide-8 and Rosa centifolia flower extract in a specific ratio, which has a synergistic effect in skin anti-aging and repair. Specifically, it can significantly synergistically reduce inflammation, promote the production of collagen I, inhibit the expression of MMP1 / MMP9 / neurotransmitter-related substance P, etc., enhance the skin's water retention capacity, improve skin elasticity, relieve and repair wrinkles, brighten the skin tone, etc.

[0041] (2) In the multilayer lipid nanoparticles provided by the present invention, palmitoyl tripeptide-8 and Rosa centifolia flower extract are loaded in different layers, thereby successfully isolating the two. In addition, the encapsulation of palmitoyl tripeptide-8 and Rosa centifolia flower extract by the multilayer lipid nanoparticles can not only significantly improve the storage stability of the active substances (especially palmitoyl tripeptide-8), but also greatly improve the transdermal properties of the active substances.

[0042] (3) The bilayer lipid middle layer of the multilayer lipid nanoparticles of the present invention is designed with a nanopore structure, which can significantly improve the suspension ability of the multilayer lipid nanoparticles, reduce sedimentation and stratification, and improve storage stability.

[0043] (4) In the preparation process of the multilayer lipid nanoparticles, the present invention strictly controls the pH so that the various layers of the multilayer lipid nanoparticles are combined through electrostatic adsorption, thereby increasing the enrichment rate and coating amount of the outer layer on the inner layer surface, and also improving the structural stability of the multilayer lipid nanoparticles themselves, thereby achieving a better encapsulation effect on the active substance and improving the stability of the active substance. DETAILED DESCRIPTION

[0044] The present invention will be further described below in conjunction with the embodiments.

[0045] Overall embodiment

[0046] A skin anti-aging and repairing composition comprises palmitoyl tripeptide-8 and Rosa centifolia flower extract in a mass ratio of 1:1-0.1:100, preferably 1:1-1:10. After separation and purification, the total content of organic acid compounds in the Rosa centifolia flower extract is at least 80wt%.

[0047] A cosmetic or skin care product with skin anti-aging and repairing effects, comprising the above-mentioned skin anti-aging and repairing composition. Preferably, the cosmetic or skin care product is a skin care product such as a face cream, lotion, water, body milk, scrub, massage cream, essence, makeup remover, cleanser, and a color makeup product such as liquid foundation, pressed powder, lipstick, and eye shadow.

[0048] A multilayer lipid nanoparticle containing palmitoyl tripeptide-8 and rosa centifolia flower extract, comprising: an aqueous core containing rosa centifolia flower extract, a double lipid middle layer coated on the surface of the aqueous core by electrostatic action and containing palmitoyl tripeptide-8 and nanopores, an aqueous middle layer containing rosa centifolia flower extract coated on the surface of the double lipid middle layer by electrostatic action, and a lipid outer layer containing palmitoyl tripeptide-8 coated on the surface of the aqueous middle layer by electrostatic action.

[0049] The preparation method of the multilayer lipid nanoparticles comprises the following steps:

[0050] (1) The Rosa centifolia flower extract is dissolved in water and glycerol, and the pH is adjusted to 5-6 to obtain a positively charged aqueous core liquid; palmitoyl tripeptide-8 is uniformly stirred with lecithin, cholesterol and ethanol aqueous solution at 40-45°C, and the pH is adjusted to 8.0-9.0 with sodium bicarbonate and sodium carbonate to obtain a negatively charged double lipid intermediate layer liquid; the negatively charged double lipid intermediate layer liquid is quickly injected into the aqueous core liquid, the ethanol is evaporated, ultrasonicated, dialyzed, freeze-dried, and heated at 50-55°C to form nanopores, thereby obtaining lipid nanoparticles A having a double lipid intermediate layer encapsulating an aqueous core structure; wherein the aqueous core liquid is loaded with the Rosa centifolia flower extract, and the double lipid intermediate layer is loaded with palmitoyl tripeptide-8. In the aqueous core liquid, the mass ratio of the Rosa centifolia flower extract, water and glycerol is 1:(85-95):(5-15). In the bilayer lipid intermediate layer liquid, the mass ratio of palmitoyl tripeptide-8, lecithin, cholesterol and ethanol aqueous solution is 1:(5-10):(2-4):(100-200); the ethanol concentration in the ethanol aqueous solution is 80-95wt%. The mass ratio of the aqueous core liquid and the bilayer lipid intermediate layer liquid is 1:(3-5).

[0051] (2) Disperse lipid nanoparticles A in anhydrous ethanol and adjust the pH to 7.5-8.0 to obtain negatively charged suspension A; dissolve Rosa centifolia flower extract in water and glycerol and adjust the pH to 3.5-4.5 to obtain a positively charged aqueous intermediate layer liquid; add suspension A dropwise to the aqueous intermediate layer liquid under stirring conditions, dialyze, and freeze-dry to obtain lipid nanoparticles B whose outermost layer is an aqueous intermediate layer; wherein the aqueous intermediate layer is loaded with Rosa centifolia flower extract. In suspension A, the mass ratio of lipid nanoparticles A to anhydrous ethanol is 1:(50-150). In the aqueous intermediate layer liquid, the mass ratio of Rosa centifolia flower extract, water, and glycerol is 1:(85-95):(5-15). The mass ratio of suspension A to aqueous intermediate layer liquid is 1:(2-3).

[0052] (3) Disperse lipid nanoparticles B in water, adjust the pH to 3.5-4.5, and obtain a positively charged suspension B; stir palmitoyl tripeptide-8, lecithin, cholesterol and anhydrous ethanol at 40-45°C, and adjust the pH to 7.5-8.0 to obtain a negatively charged lipid outer layer liquid; add suspension B dropwise to the lipid outer layer liquid under stirring conditions, dialyze, and freeze-dry to obtain multilayer lipid nanoparticles (particle size 50-100nm) with the outermost layer being the lipid outer layer; wherein the lipid outer layer is loaded with palmitoyl tripeptide-8. In suspension B, the mass ratio of lipid nanoparticles B to water is 1:(50-150). In the lipid outer layer liquid, the mass ratio of palmitoyl tripeptide-8, lecithin, cholesterol and anhydrous ethanol is 1:(5-10):(2-4):(100-200). The mass ratio of suspension B to lipid outer layer liquid is 1:(2-4).

[0053] Examples 1-5 and Comparative Examples 1-2

[0054] In order to study the compounding effect of palmitoyl tripeptide-8 and Rosa centifolia flower extract, active solution (using glycerol as solvent) was prepared according to the formula of Examples 1-5 and Comparative Examples 1-2 in the following table. The source of palmitoyl tripeptide-8 is (Neutrazen, Lucas Meyer), and the source of Rosa centifolia flower extract is (StandStill Rose from Damas, Naolys, the total amount of organic acid after separation and purification is about 90wt%).

[0055] Test Group A: Palmitoyl Tripeptide-8 concentration B: Concentration of Rosa centifolia flower extract Mass ratio (A / B) Example 1 0.1wt% 0.01wt% 1∶0.1 Example 2 0.1wt% 0.1wt% 1∶1 Example 3 0.1wt% 1.0wt% 1∶10 Example 4 0.1wt% 5.0wt% 1∶50 Example 5 0.1wt% 10.0wt% 1：100 Comparative Example 1 0.1wt% 0wt% / Comparative Example 2 0wt% 10.0wt% /

[0056] Effect test

[0057] The effects of the active substance solutions prepared in Examples 1-5 and Comparative Examples 1-2 were tested as follows:

[0058] (1) Skin irritation test: About 24 hours before the test, the hair on both sides of the spine of the back of SD rats (8 weeks old, male) was cut off without damaging the epidermis. The hair removal range was about 3 cm×3 cm on the left and right. About 0.5 mL of the active solution of Examples 1-5 and Comparative Examples 1-2 was applied directly to the skin, and then covered with two layers of gauze (2.5 cm×2.5 cm) and a layer of cellophane, and then fixed with non-irritating adhesive tape and bandage. The skin on the other side was used as a control. A closed test was used, and the application time was 4 hours. After the test, the residual test substance was removed with warm water. The skin reaction of the application site was observed 1 hour, 24 hours, 48 ​​hours and 72 hours after the removal of the test substance, and the skin reaction score was performed according to the table below. The average value of the scores of the test animals was used for comprehensive evaluation. The skin irritation intensity was determined based on the average value of the highest score at each observation time point of 24 hours, 48 ​​hours and 72 hours.

[0059] Skin irritation intensity classification

[0060]

[0061]

[0062] Results of skin irritation test on SD rats

[0063]

[0064] Conclusion: The active substance solutions of Examples 1-5 and Comparative Examples 1-2 have no acute skin irritation to SD rats.

[0065] (2) Efficacy test (2.1) Test for the ability to inhibit inflammatory factors: RAW264.7 cells in the logarithmic growth phase were taken and the cell density was adjusted to 5×105 Cells were inoculated into multi-well plates. The cells were divided into 9 groups, as shown in the following table:

[0066] Grouping Processing Normal control group Add 2 mL of cell culture medium LPS intervention group Treatment with 1 μg / mL LPS without active substance intervention Example 1 After pretreatment with the active substance solution of Example 1 for 2 h, 1 μg / mL LPS was added for intervention for 24 h. Example 2 After pretreatment with the active substance solution of Example 2 for 2 h, 1 μg / mL LPS was added for intervention for 24 h. Example 3 After pretreatment with the active substance solution of Example 3 for 2 h, 1 μg / mL LPS was added for intervention for 24 h. Example 4 After pretreatment with the active substance solution of Example 4 for 2 h, 1 μg / mL LPS was added for intervention for 24 h. Example 5 After pretreatment with the active substance solution of Example 5 for 2 h, 1 μg / mL LPS was added for intervention for 24 h. Comparative Example 1 After pretreatment with the active substance solution of Comparative Example 1 for 2 h, 1 μg / mL LPS was added for intervention for 24 h. Comparative Example 2 After pretreatment with the active substance solution of Comparative Example 2 for 2 h, 1 μg / mL LPS was added for intervention for 24 h.

[0067] After incubation for 24 h, the contents of NO, TNF-α, and IL-6 in the supernatant were detected by flow cytometry. The results are shown in the following table.

[0068]

[0069]

[0070] As can be seen from the data in the above table, the secretion of NO, TNF-α, and IL-6 in the normal control group is at a low level. When LPS is stimulated in the LPS intervention group, the secretion of cellular NO, IL-6, and TNF-α increases significantly, with significant differences. In Examples 1-5 and Comparative Example 1, after being treated with active substances compounded in different proportions before LPS intervention, the secretion of cellular NO, IL-6, and TNF-α is significantly lower than that of the LPS intervention group. The results show that the active substances in Examples 1-5 and Comparative Example 1 can inhibit the expression level of proinflammatory factors and have significant anti-inflammatory effects. The anti-inflammatory effect of pure Rosa centifolia flower extract in Comparative Example 2 is not obvious, but Examples 1-5 use a single palmitoyl tripeptide-8 compared to Comparative Example 1, and the anti-inflammatory ability of the two after compounding is stronger. And when the concentration ratio of palmitoyl tripeptide-8 to Rosa centifolia flower extract reaches 1:10 (Example 3), the concentration of Rosa centifolia flower extract is continued to be increased, and the improvement of the anti-inflammatory effect gradually slows down.

[0071] (2.2) Test on the effect of promoting the synthesis of collagen-related proteins: 8 experimental groups (blank control group, Examples 1-5 and Comparative Examples 1-2) were set up, and the proliferated cells (normal human skin fibroblasts) were dispersed in Dulbecco'sMEM medium containing FBS and inoculated in a multi-well culture plate. Then, the cells were cultured in a CO2 incubator (37°C). On the second day after inoculation, the culture medium was replaced with Dulbecco's MEM medium containing FBS added with active solution of Examples 1-5 and Comparative Examples 1-2 (the added amount was 50wt%) and the blank group, and then cultured in a CO2 incubator. Afterwards, the supernatant was collected and the amount of type I collagen was determined by ELISA. After collecting the supernatant, the amount of type I collagen per unit protein was calculated by lysing the cells, and the results were deduced and evaluated. The results are shown in the following table.

[0072] Grouping Type I collagen content (μg / mL) Blank control group 17.45 Example 1 29.15 Example 2 31.67 Example 3 33.45 Example 4 33.89 Example 5 34.12 Comparative Example 1 19.12 Comparative Example 2 23.41

[0073] As can be seen from the data in the above table, the results show that compared with the blank control group, the active substance solutions of Examples 1-5 and Comparative Example 1 can promote the production of type I collagen, among which the effect of Examples 1-5 is better than that of Comparative Example 1-2, indicating that the compound of palmitoyl tripeptide-8 and Rosa centifolia flower extract is better than the single ingredient.

[0074] (2.3) Test of the inhibitory ability of MMP1 and MMP9: Human skin fibroblasts were placed in a culture bottle under sterile conditions and cultured in a 37°C, 5% CO2 incubator. When the cells covered about 70% of the bottle wall, subculture was performed. Take the 3rd to 5th generation cells. The 3rd to 5th generation cells were divided into a control group, a model group and 7 experimental groups (Examples 1-5 and Comparative Examples 1-2), wherein: 200 μL of serum-free DMEM was added to the control group and cultured for 24 hours; the model group cells were cultured for 24 hours and then irradiated with ultraviolet light, and 200 μL of DMEM was given; the experimental group cells were cultured for 24 hours, and after ultraviolet irradiation, 200 μL of the active solution of Examples 1-5 and Comparative Examples 1-2 were added respectively. After 24 hours of action, the MMP-1 and MMP-9 contents of each group of samples were determined by double antibody sandwich ELISA, and the sample absorbance value was determined at 450nm using an enzyme marker. The sample concentration was calculated based on the sample absorbance value. The concentration data are shown in the following table:

[0075] Group MMP-1 concentration (pg / mL) MMP-9 concentration (pg / mL) Control group 582.16 572.78 Model Group 987.65 932.41 Example 1 759.21 752.31 Example 2 709.74 715.22 Example 3 654.73 668.54 Example 4 633.62 637.65 Example 5 598.65 619.24 Comparative Example 1 869.43 843.58 Comparative Example 2 647.34 694.36

[0076] The lower the concentration of MMP-1 and MMP-9 proteins, the stronger the cell antioxidant capacity. It can be seen from the data in the above table that compared with the control group, the expression levels of MMP1 and MMP9 in the model group increased significantly, indicating that the modeling was successful. Compared with the control group, the concentrations of MMP1 and MMP9 in Comparative Example 1 did not change much, indicating that the inhibitory effect of a single palmitoyl tripeptide-8 on MMP1 and MMP9 was not ideal. Compared with the model group, both indicators of Examples 1-5 and Comparative Example 2 decreased significantly, indicating that Examples 1-5 and Comparative Example 2 can significantly inhibit MMP1 and MMP9. In addition, according to the data trend, it can be found that the appropriate proportion of palmitoyl tripeptide-8 and Rosa centifolia flower extract can significantly enhance the inhibitory ability of Rosa centifolia flower extract on MMP1 and MMP9, but when the concentration ratio of Rosa centifolia flower extract reaches 1:10 (Example 3), the concentration of Rosa centifolia flower extract is continued to be increased, and the effect of the improvement gradually slows down.

[0077] (2.4) Inhibitory ability of substance P related to neurotransmitters: 54 male 10-week-old SD rats weighing 250-280 g were prepared. The 54 rats were randomly divided into 9 groups, including a control group, a model group, and an experimental group (Examples 1-5 and Comparative Examples 1-2), with 6 rats in each group. The sciatic nerve under the gluteus maximus muscle of the right thigh of the rats in the model group and the experimental group was exposed, and the right sciatic nerve was loosely ligated at 3 points with No. 1 non-absorbable braided silk thread. The sciatic nerve was completely ligated with the thread knot without notches. The pain test determined that the pain reached the peak on the 15th day of modeling. After the pain test on the 15th day of modeling, the 7 experimental groups were given 10 mg / kg of the active substance solution of Examples 1-5 and Comparative Examples 1-2 by intramuscular injection in the right thigh, and the control group was not given any medicine. After the 15th day of modeling, the 9 groups of rats were killed by cervical dislocation, and the skin specimens of the dorsal foot on the same side of the ligation were taken, and pathological sections were prepared for immunohistochemical detection. The SD rat skin samples were immediately separated using sterile instruments and frozen at -80°C for future use in mRNA determination.

[0078] SP protein level was detected by immunohistochemistry. The skin of the right dorsum of the rats in each group was cut into slices. After drying, dewaxing, hydration, rinsing, endogenous enzyme blocking, and blocking specific antigens, the primary antibody of SP was added overnight. The slices were taken out and rinsed the next day, and then 1:200 secondary antibodies were added for incubation and rinsing. After staining, the slices were sealed with neutral gum. Image-program 5.0 image analysis software was used to analyze the distribution index of SP immunohistochemistry, and the cumulative absorbance (D) value of the brown substance in the skin basal cells in the 200x image field under the microscope was counted.

[0079] SP mRNA detection: RT-PCR detection was performed after extracting 1g of mRNA from the dorsal skin of the foot. β-actin (Sigma, USA) was used as the internal reference, and the primer sequence was: SP upstream primer 5′-ATGAAAATCCTCGTGGCGGT-3′, downstream primer 5′-CAGCATCCCGTTTGCCCATT-3′, and the product was 219bp. The D value was measured and the ratio of the gray value of the target band to the gray value of the internal reference electrophoresis band was calculated.

[0080] The data of the above tests are shown in the following table:

[0081]

[0082] The neurotransmitter substance P (also known as SP substance) is the earliest neuropeptide discovered in the world. It supports the SP-positive sensory neurons of the skin to have a bidirectional conduction function. The SP released by it can not only transmit stimulation information to the central nervous system as a neurotransmitter, but also can be a neuromodulator to exert biological effects in the periphery, playing an important role in skin immune inflammation, wound repair, itching, etc. The lower the expression level of substance P, the lower the level of cellular inflammation. It can be seen from the data in the above table that compared with the control group, the protein level of substance P and the expression level of substance P in the model group increased significantly, indicating that the modeling was successful. Compared with the model group, the two indicators of comparative example 2 did not change much, indicating that a single extract of Rosa centifolia flower had almost no effect on the inhibition of substance P. Compared with the model group, the two indicators of Examples 1-5 and Comparative Example 1 decreased significantly, indicating that Examples 1-5 and Comparative Example 1 can significantly inhibit the expression of substance P. Moreover, according to the data trend, it can be found that the appropriate ratio of the compound of Rosa centifolia flower extract and palmitoyl tripeptide-8 can enhance the ability of palmitoyl tripeptide-8 to inhibit substance P. When the concentration ratio of Rosa centifolia flower extract reaches 1:10 (Example 3), the concentration of Rosa centifolia flower extract continues to increase, and the effect improvement gradually slows down.

[0083] Example 6 (Palmitoyl Tripeptide-8 + Rosa Centifolia Flower Extract)

[0084] (1) Dissolve 1 part of Rosa centifolia flower extract (total organic acid of about 90 wt%) in 90 parts of water and 10 parts of glycerol, adjust the pH to 5.5 with dilute hydrochloric acid, and obtain a positively charged aqueous core liquid; stir 1 part of palmitoyl tripeptide-8 with 7.5 parts of lecithin, 3 parts of cholesterol, and 150 parts of 90 wt% ethanol aqueous solution at 40°C, and adjust the pH to 8.5 with sodium bicarbonate and sodium carbonate to obtain a negatively charged bilayer lipid intermediate layer liquid. Rapidly inject the bilayer lipid intermediate layer liquid into the aqueous core liquid at a mass ratio of 4:1, evaporate the ethanol, perform ultrasound, dialyze, freeze-dry, and heat at 55°C to form nanopores to obtain lipid nanoparticles A.

[0085] (2) Disperse 1 part of lipid nanoparticle A in 100 parts of anhydrous ethanol, adjust the pH to 7.8 with sodium hydroxide to obtain a negatively charged suspension A; dissolve 1 part of Rosa centifolia flower extract in 90 parts of water and 10 parts of glycerol, adjust the pH to 4.0 with dilute hydrochloric acid to obtain a positively charged aqueous intermediate layer liquid; under stirring conditions, add the suspension A dropwise to the aqueous intermediate layer liquid at a mass ratio of 1:2.5, dialyze, and freeze-dry to obtain lipid nanoparticle B.

[0086] (3) Disperse 1 part of lipid nanoparticle B into 100 parts of water, adjust the pH to 4.0 with dilute hydrochloric acid to obtain a positively charged suspension B; stir 1 part of palmitoyl tripeptide-8 with 7.5 parts of lecithin, 3 parts of cholesterol and 150 parts of anhydrous ethanol at 40°C, adjust the pH to 7.8 with sodium hydroxide to obtain a negatively charged lipid outer layer liquid; under stirring, add the suspension B dropwise to the lipid outer layer liquid at a mass ratio of 1:3, dialyze, and freeze-dry to obtain multilayer lipid nanoparticles.

[0087] Comparative Example 3 (Carrier is ordinary liposome)

[0088] Dissolve 1 part of Rosa centifolia flower extract (about 90 wt% of total organic acid) in 90 parts of water and 10 parts of glycerol, adjust the pH to 4.0 with dilute hydrochloric acid to obtain a positively charged aqueous core liquid; stir 1 part of palmitoyl tripeptide-8 with 7.5 parts of lecithin, 3 parts of cholesterol and 150 parts of anhydrous ethanol at 40°C, and adjust the pH to 7.8 with sodium hydroxide to obtain a negatively charged double-layer lipid outer liquid. Quickly inject the double-layer lipid outer liquid into the aqueous core liquid at a mass ratio of 4:1, evaporate the ethanol, sonicate, dialyze, and freeze-dry to obtain liposome particles.

[0089] Comparative Example 4 (Multilayer lipid nanoparticles are not electrostatically bound to each other)

[0090] (1) Dissolve 1 part of Rosa centifolia flower extract (total organic acid of about 90 wt%) in 90 parts of water and 10 parts of glycerol, adjust the pH to neutral, and obtain an aqueous core liquid; stir 1 part of palmitoyl tripeptide-8 with 7.5 parts of lecithin, 3 parts of cholesterol, and 150 parts of 90 wt% ethanol aqueous solution at 40°C, and adjust the pH to neutral to obtain a lipid intermediate layer liquid. Rapidly inject the double lipid intermediate layer liquid into the aqueous core liquid at a mass ratio of 4:1, evaporate the ethanol, sonicate, dialyze, and freeze-dry to obtain lipid nanoparticles A.

[0091] (2) Disperse 1 part of lipid nanoparticle A in 100 parts of anhydrous ethanol, adjust the pH to neutral, and obtain suspension A; dissolve 1 part of Rosa centifolia flower extract in 90 parts of water and 10 parts of glycerol, adjust the pH to neutral, and obtain an aqueous intermediate layer liquid; under stirring conditions, add the suspension A dropwise to the aqueous intermediate layer liquid at a mass ratio of 1:2.5, dialyze, and freeze-dry to obtain lipid nanoparticle B.

[0092] (3) Disperse 1 part of lipid nanoparticle B into 100 parts of water, adjust the pH to neutral, and obtain suspension B; stir 1 part of palmitoyl tripeptide-8 with 7.5 parts of lecithin, 3 parts of cholesterol, and 150 parts of anhydrous ethanol at 40°C, adjust the pH to neutral, and obtain a lipid outer layer liquid; under stirring, add suspension B dropwise into the lipid outer layer liquid at a mass ratio of 1:3, dialyze, and freeze-dry to obtain multilayer lipid nanoparticles.

[0093] Comparative Example 5 (Multilayer lipid nanoparticles without nanopores)

[0094] (1) Dissolve 1 part of Rosa centifolia flower extract (total organic acid content of about 90 wt%) in 90 parts of water and 10 parts of glycerol, and adjust the pH to 4.0 with dilute hydrochloric acid to obtain a positively charged aqueous core liquid; stir 1 part of palmitoyl tripeptide-8 with 7.5 parts of lecithin, 3 parts of cholesterol, and 150 parts of 90 wt% ethanol aqueous solution at 40°C, and adjust the pH to 7.8 with sodium hydroxide to obtain a negatively charged bilayer lipid intermediate layer liquid. Rapidly inject the bilayer lipid intermediate layer liquid into the aqueous core liquid at a mass ratio of 4:1, evaporate the ethanol, perform ultrasound, dialyze, and freeze-dry to obtain lipid nanoparticles A.

[0095] (2) Preparation of lipid nanoparticles B: same as Example 6.

[0096] (3) Preparation of multilayer lipid nanoparticles: same as Example 6.

[0097] Comparative Example 6 (pH of each intermediate solution or suspension is different)

[0098] (1) Dissolve 1 part of Rosa centifolia flower extract (total organic acid content of about 90 wt%) in 90 parts of water and 10 parts of glycerol, adjust the pH to 6.5 with dilute hydrochloric acid, and obtain an aqueous core liquid; stir 1 part of palmitoyl tripeptide-8 with 7.5 parts of lecithin, 3 parts of cholesterol, and 150 parts of 90 wt% ethanol aqueous solution at 40°C, and adjust the pH to 7.5 with sodium bicarbonate and sodium carbonate to obtain a bilayer lipid intermediate layer liquid. Rapidly inject the bilayer lipid intermediate layer liquid into the aqueous core liquid at a mass ratio of 4:1, evaporate the ethanol, perform ultrasound, dialyze, freeze-dry, and heat at 55°C to form nanopores to obtain lipid nanoparticles A.

[0099] (2) Disperse 1 part of lipid nanoparticle A in 100 parts of anhydrous ethanol, adjust the pH to 7.2 with sodium hydroxide to obtain suspension A; dissolve 1 part of Rosa centifolia flower extract in 90 parts of water and 10 parts of glycerol, adjust the pH to 6.5 with dilute hydrochloric acid to obtain an aqueous intermediate layer liquid; add the suspension A dropwise to the aqueous intermediate layer liquid at a mass ratio of 1:2.5 under stirring, dialyze, and freeze-dry to obtain lipid nanoparticle B.

[0100] (3) Disperse 1 part of lipid nanoparticle B into 100 parts of water, adjust the pH to 6.5 with dilute hydrochloric acid to obtain suspension B; stir 1 part of palmitoyl tripeptide-8 with 7.5 parts of lecithin, 3 parts of cholesterol and 150 parts of anhydrous ethanol at 40°C, adjust the pH to 7.2 with sodium hydroxide to obtain a negatively charged lipid outer layer liquid; under stirring, add suspension B dropwise to the lipid outer layer liquid at a mass ratio of 1:3, dialyze, and freeze-dry to obtain multilayer lipid nanoparticles.

[0101] Performance Testing

[0102] (1) Stability test: The liposome particles or multilamellar lipid nanoparticles prepared in Example 6 and Comparative Examples 3-6 were formulated into emulsions (concentration 5 wt%) and then subjected to stability test, as follows: The preparations prepared in Example 6 and Comparative Examples 3-6 were left to stand at 25° C. for 90 days, and the results were shown in the following table:

[0103]

[0104]

[0105] As can be seen from the data in the above table, the average particle size of the emulsion containing the multilayer lipid nanoparticles of Example 6 changed little after 90 days, and the emulsion remained uniform throughout, without stratification, sedimentation, agglomeration, and precipitation, indicating good stability. The emulsion containing the liposome particles of Comparative Example 3 performed well in terms of particle size stability within 60 days, with the particle size only slightly increasing, but the average particle size increased significantly after 90 days, and slight agglomeration occurred. The emulsion containing the multilayer lipid nanoparticles of Comparative Example 4 (due to the fact that the nanoparticles were not electrostatically bonded between the layers during the preparation process, the The interlayers are not dense enough, the particle size is too large, and the particle size increases significantly during storage, resulting in a more serious agglomeration phenomenon, and stratification and sedimentation are also prone to occur. The emulsion containing multilayer lipid nanoparticles in comparative example 5 (is superior in particle size stability, but due to the nanopores contained in the nanoparticles, the suspension ability is poor, and sedimentation and stratification gradually occur after 60 days. The emulsion containing multilayer lipid nanoparticles in comparative example 5 (due to improper pH regulation of the nanoparticles during the preparation process, the particle size is out of control, and gradually agglomerates and precipitates after 60 days.

[0106] (2) Skin irritation test: About 24 hours before the test, the hair on both sides of the back spine of SD rats (8 weeks old, male) was trimmed without damaging the epidermis. The hair removal range was about 3 cm×3 cm on the left and right sides. The emulsions of Example 6 and Comparative Example 3 (about 0.5 mL) were applied directly to the skin, and then covered with two layers of gauze (2.5 cm×2.5 cm) and a layer of cellophane, and then fixed with non-irritating adhesive tape and bandage. The skin on the other side was used as a control. A closed test was used, and the application time was 4 hours. After the test, the residual test substance was removed with warm water. The skin reaction of the applied area was observed 1 hour, 24 hours, 48 ​​hours and 72 hours after the removal of the test substance. The skin reaction score was calculated according to the table below. The average value of the scores of the test animals was used for comprehensive evaluation. The skin irritation intensity was determined based on the average value of the highest score at each observation time point of 24 hours, 48 ​​hours and 72 hours.

[0107] Skin irritation intensity classification

[0108] Integral mean strength 0～＜0.5 Non-irritating 0.5～＜2.0 Mild irritation 2.0～＜6.0 Moderate irritation 6.0～8.0 Strong irritation

[0109] Results of skin irritation test on SD rats

[0110]

[0111]

[0112] Conclusion: The preparations of Example 6 and Comparative Example 3 had no acute skin irritation to SD rats.

[0113] (3) Human testing:

[0114] 40 healthy Chinese female subjects who considered themselves to have dry and rough facial skin, loose and inelastic facial skin, and obvious fine lines on the face were selected, with an age range of 40-50 years old, and were evenly divided into 2 groups. Each group used the emulsion of Example 6 and Comparative Example 3 and the blank control method, and the before-after control method was used to test the transepidermal water loss value and skin elasticity of the test site of the subjects before using the emulsion, and after using the emulsion for 14 days and 28 days (Note: the ambient temperature during the test was 20.0-25.0°C and the relative humidity was 45-60%RH).

[0115]

[0116] The test results are as follows:

[0117] (3.1) Detection results of the area ratio of eye wrinkles:

[0118] Grouping Base value (D0) Use 14 day value Improvement rate after 14 days of use Use 28 day value Improvement rate after 28 days of use Example 6 16.78％ 14.58％ 13.11％ 13.16％ 21.57％ Comparative Example 3 16.22％ 15.33％ 5.49％ 14.47％ 10.78％

[0119] As can be seen from the data in the above table, the emulsions of Example 6 and Comparative Example 3 can reduce eye wrinkles to a certain extent. Compared with Comparative Example 3, Example 6 has a better effect, which indicates that the multilayer lipid nanoparticles of Example 6 have better skin permeability than ordinary liposomes, and can be fully absorbed by the skin.

[0120] (3.2) Skin color L* value test results:

[0121] Grouping Base value (D0) Use 14 day value Improvement rate after 14 days of use Use 28 day value Improvement rate after 28 days of use Example 6 90.15 90.47 0.35％ 90.81 0.73％ Comparative Example 3 90.21 90.38 0.19％ 90.59 0.42％

[0122] As can be seen from the data in the above table, the emulsions of Example 6 and Comparative Example 3 can both improve the L* value of skin color to a certain extent. The higher the value, the whiter the skin color. Among them, the effect of Example 6 is better, which shows that the multilayer lipid nanoparticles of Example 6 have better skin permeability than ordinary liposomes, and can be fully absorbed by the skin.

[0123] (3.3) Skin color b* value detection results:

[0124] Grouping Base value (D0) Use 14 day value Improvement rate after 14 days of use Use 28 day value Improvement rate after 28 days of use Example 6 12.93 12.60 2.56％ 12.33 4.64％ Comparative Example 3 12.88 12.69 1.48％ 12.49 3.03％

[0125] As can be seen from the data in the above table, the emulsions of Example 6 and Comparative Example 3 can reduce the b* value of skin color to a certain extent. The lower the value, the less yellow the skin color. Among them, the effect of Example 6 is better, which shows that the multilayer lipid nanoparticles of Example 6 have better skin permeability than ordinary liposomes, and can be fully absorbed by the skin.

[0126] (3.4) Skin color ITA° value test results:

[0127] Grouping Base value (D0) Use 14 day value Improvement rate after 14 days of use Use 28 day value Improvement rate after 28 days of use Example 6 72.01 72.63 0.86％ 72.99 1.36％ Comparative Example 3 72.10 72.42 0.44％ 72.71 0.85％

[0128] As can be seen from the data in the above table, the emulsions of Example 6 and Comparative Example 3 can both improve the ITA° value of skin color to a certain extent. The greater the GAP of this value, the lighter the skin color. Among them, the effect of Example 6 is better, which explains that the multilayer lipid nanoparticles of Example 6 have better skin permeability than ordinary liposomes, and can be fully absorbed by the skin.

[0129] (3.5) Skin transepidermal water loss test:

[0130] Grouping Improvement rate after 14 days of use Improvement rate after 28 days of use Example 6 26.49％ 33.33％ Comparative Example 3 21.78％ 28.57％

[0131] As can be seen from the data in the above table, both in the short-term / long-term and skin transepidermal water loss tests, compared with the blank control group, Example 6 and Comparative Example 3 can significantly improve the skin's moisturizing ability. Among them, Example 6 has a better effect, which explains that the multilayer lipid nanoparticles of Example 6 have better skin permeability than ordinary liposomes, and can be fully absorbed by the skin.

[0132] (3.6) Skin elasticity R5 test:

[0133]

[0134] As can be seen from the data in the above table, the emulsions of Example 6 and Comparative Example 3 can improve skin elasticity to a certain extent, and thus have good anti-wrinkle repairing ability. Among them, the effect of Example 6 is better, which shows that the multilayer lipid nanoparticles of Example 6 have better skin permeability than ordinary liposomes, and can be fully absorbed by the skin.

[0135] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0136] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing multi-layer lipid nanoparticles, characterized in that: 1) Dissolve the Rosa centifolia flower extract in water and glycerol, adjust the pH to 5-6, and obtain a positively charged aqueous core liquid; stir palmitoyl tripeptide-8 with lecithin, cholesterol and ethanol aqueous solution at 40-45°C, and adjust the pH to 8-9 with sodium bicarbonate and sodium carbonate to obtain a negatively charged bilayer lipid intermediate liquid; The bilayer lipid intermediate layer liquid is quickly injected into the aqueous core liquid, ethanol is evaporated, ultrasonication is performed, dialysis is performed, freeze-drying is performed, and heating is performed at 50-55° C. to form nanopores, thereby obtaining lipid nanoparticles A with a bilayer lipid intermediate layer covering an aqueous core liquid; the aqueous core liquid is loaded with a Rosa centifolia flower extract, and the bilayer lipid intermediate layer is loaded with palmitoyl tripeptide-8; 2) Dispersing lipid nanoparticles A in anhydrous ethanol, adjusting the pH to 7.5-8, to obtain a negatively charged suspension A; dissolving the Rosa centifolia flower extract in water and glycerol, adjusting the pH to 3.5-4.5, to obtain a positively charged aqueous intermediate layer liquid; The suspension A is dripped into the aqueous intermediate layer liquid under stirring, dialyzed, and freeze-dried to obtain lipid nanoparticles B whose outermost layer is the aqueous intermediate layer; the aqueous intermediate layer is loaded with the Rosa centifolia flower extract; 3) Disperse lipid nanoparticles B in water, adjust the pH to 3.5-4.5, and obtain a positively charged suspension B; stir palmitoyl tripeptide-8, lecithin, cholesterol and anhydrous ethanol at 40-45°C, adjust the pH to 7.5-8, and obtain a negatively charged lipid outer layer liquid; drop the suspension B into the lipid outer layer liquid under stirring, dialyze, and freeze-dry to obtain multilayer lipid nanoparticles whose outermost layer is the lipid outer layer; the lipid outer layer is loaded with palmitoyl tripeptide-8; The mass ratio of palmitoyl tripeptide-8 to the Rosa centifolia flower extract is 1:0.1-100; the content of organic acid compounds in the Rosa centifolia flower extract is ≥80wt%.

2. The preparation method according to claim 1, characterized in that: The mass ratio of the palmitoyl tripeptide-8 to the Rosa centifolia flower extract is 1:1-10.

3. The preparation method according to claim 1, characterized in that: In step 1): In the aqueous phase inner core liquid, the mass ratio of Rosa centifolia flower extract, water and glycerin is 1: (85-95): (5-15); In the bilayer lipid intermediate layer liquid, the mass ratio of palmitoyl tripeptide-8, lecithin, cholesterol and ethanol aqueous solution is 1: (5-10): (2-4): (100-200); the ethanol concentration in the ethanol aqueous solution is 80-95wt%; The mass ratio of the inner core liquid in the aqueous phase to the bilayer lipid middle layer liquid is 1:(3-5).

4. The preparation method according to claim 1, characterized in that: In step 2): In the suspension A, the mass ratio of lipid nanoparticles A to anhydrous ethanol is 1:(50-150); In the aqueous intermediate layer liquid, the mass ratio of Rosa centifolia flower extract, water and glycerin is 1: (85-95): (5-15); The mass ratio of the suspension A to the aqueous intermediate layer liquid is 1:(2-3).

5. The preparation method according to claim 1, characterized in that: In step 3): In the suspension B, the mass ratio of lipid nanoparticles B to water is 1:(50-150); In the lipid outer layer liquid, the mass ratio of palmitoyl tripeptide-8, lecithin, cholesterol and anhydrous ethanol is 1: (5-10): (2-4): (100-200); The mass ratio of the suspension B to the lipid outer layer liquid is 1:(2-4).

6. The preparation method according to claim 1, characterized in that: The particle size of the multi-layer lipid nanoparticles is 50-100 nm.

7. A multi-layer lipid nanoparticle obtained by the preparation method according to any one of claims 1 to 6, characterized in that: include: Contains an aqueous core containing Rosa centifolia flower extract, A double lipid intermediate layer including palmitoyl tripeptide-8 and nanopores is coated on the surface of the aqueous core by electrostatic action, The aqueous phase intermediate layer containing the Rosa centifolia flower extract is coated on the surface of the bilayer lipid intermediate layer by electrostatic action, The lipid outer layer comprising palmitoyl tripeptide-8 is coated on the surface of the aqueous phase intermediate layer through electrostatic action.

8. Use of the multilayer lipid nanoparticles according to claim 7 in cosmetics or skin care products with skin anti-aging and repairing effects.

9. The use according to claim 8, characterized in that: The cosmetics or skin care products are face cream, lotion, scrub, massage cream, make-up remover cream, pressed powder, lipstick or eye shadow.

10. The use according to claim 8, characterized in that: The cosmetics or skin care products are essence, cleansing milk or liquid foundation.

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