Nanometer lipid aggregates with bionic double-periodic layered structure, and preparation method and application thereof

Abstract

The application discloses a kind of nano lipid aggregate with bionic double periodic layered structure and its preparation method and application, belong to skin care technical field.The nano lipid aggregate of the application has the double periodic layered structure of bionic short periodic phase+long periodic phase, which is formed by embedding rare ginsenoside CK in its inside by lipid bilayer;And the molar ratio of long periodic phase in lipid bilayer is >60%.The nano lipid aggregate with the double periodic layered structure of bionic short periodic phase+long periodic phase is obtained by compounding specific component and matching ratio of composite ceramide with rare ginsenoside CK, and combining temperature control emulsification process.The nano lipid aggregate can realize the dual enhancement of skin barrier repair and anti-aging effect, and simultaneously efficiently meet the demand of comprehensive barrier repair and anti-aging.Therefore, the nano lipid aggregate of the application has good application prospect.

Description

Technical Field

[0001] This invention relates to the field of skincare technology, and more specifically, to a nanolipid aggregate with a biomimetic dual-period layered structure, its preparation method, and its application. Background Technology

[0002] Ceramides are a core component of the intercellular lipids in the stratum corneum of the skin. Together with cholesterol and free fatty acids, they form a dense bilayer structure, constituting the most important physical barrier for the human skin. This barrier function not only effectively reduces the evaporation of water from within the skin (i.e., reduces transepidermal water loss) and maintains stratum corneum hydration, keeping the skin moisturized and elastic, but also resists the invasion of external irritants and pathogens. Simultaneously, ceramides, as bioactive molecules, participate in regulating physiological processes such as the proliferation, differentiation, and apoptosis of keratinocytes, which are crucial for maintaining healthy epidermal renewal. When the ceramide content in the skin decreases or the ratio of various subtypes becomes imbalanced, the skin barrier is damaged, leading to dryness, sensitivity, and even triggering skin diseases such as atopic dermatitis. Therefore, many repair-oriented skincare products add ceramides to achieve skin barrier repair.

[0003] Ginsenosides are the main active ingredients of ginseng plants, belonging to the triterpenoid glycoside class of compounds. Based on their aglycone structures, they can be classified into protopanaxadiol, protopanatriol, and oleanane types, among others, with hundreds identified to date. In skin care, modern pharmacological studies have shown that ginsenosides exhibit various skin-care effects both in vitro and in vivo, including anti-aging, sun protection, whitening, moisturizing, and anti-inflammatory properties. Some rarer ginsenosides (such as Rh1 and CK) have attracted attention due to their better bioavailability. Because of their multiple biological activities, ginsenosides have become an important raw material for anti-aging and skin care.

[0004] Combining active ingredients with different functions to achieve more comprehensive skincare effects is a common approach in skincare product design. However, ceramides and ginsenosides have fundamentally contradictory physicochemical properties: ceramides are lipid-soluble macromolecules (molecular weight approximately 540-650), requiring dissolution in the oil phase and dispersion via an emulsion system; ginsenoside CK is a water-soluble, moderately polar molecule (molecular weight 622.88), requiring dissolution in the aqueous phase. Currently, the common method for combining these two types of ingredients in the same formulation is as follows: ceramides are dissolved at high temperature and added to the oil phase, then emulsified with the aqueous phase to form an emulsion. After the emulsion cools, an aqueous solution of ginsenosides is added. In this process, the two types of ingredients are independently dispersed in the oil and aqueous phases of the emulsion system, respectively, without structural interaction. This significantly reduces the overall skincare effect of the resulting product and affects its stability.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a biomimetic double-periodic layered structure of nanolipid aggregates, its preparation method, and its application. The nanolipid aggregates are formed by in-situ self-assembly of an oil phase prepared from a composite ceramide with a specific composition and lipid components with an aqueous phase prepared from rare ginsenoside CK. The rare ginsenoside CK is spontaneously positioned at the water-lipid interface of the lipid bilayer, achieving in-situ co-assembly and embedding. The obtained nanolipid aggregates produce a synergistic effect in skin care.

[0007] This invention is implemented as follows: In a first aspect, the present invention provides a nanolipid aggregate with a biomimetic dual-period layered structure, which has a biomimetic short-period phase + long-period phase dual-period layered structure, and is formed by embedding rare ginsenoside CK in a lipid bilayer. The long-period phase accounts for >60% of the molar proportion in the lipid bilayer; the lipid bilayer is composed of complex ceramides and lipid components; the complex ceramides include ceramide subtypes NP, AP, EOP, and EOS, with a mass ratio of 10:2.5:1:1; the mass ratio of water-soluble complex ceramides to rare ginsenoside CK is 0.5~5:0.01~1.

[0008] In a second aspect, the present invention provides a method for preparing the above-mentioned nanolipid aggregates, comprising: The composite ceramide and lipid components were mixed and stirred continuously at 95°C until completely dissolved and in a uniform and transparent state to obtain the oil phase; water was heated to 55~70°C, and a thickener and a pre-solution of rare ginsenoside CK were added and mixed to prepare the aqueous phase; the oil phase was slowly added to the aqueous phase, and after homogenization and emulsification, the in-situ self-assembly of nanolipid aggregates was completed.

[0009] Thirdly, the present invention provides the application of the above-mentioned nanolipid aggregates or nanolipid aggregates prepared by the above-mentioned preparation method in the preparation of skin care products.

[0010] Fourthly, the present invention provides a skin care product comprising the above-mentioned nanolipid aggregates; the skin care product comprises the following raw materials by mass percentage: 0.5%-5% compound ceramide, 0.01%-1% rare ginsenoside CK, 0.5%-5% cholesterol, 0.5%-3% stearic acid, 1%-5% squalane, and 0.1%-0.5% thickener.

[0011] The present invention has the following beneficial effects: This invention obtains nanolipid aggregates with a biomimetic short-period phase + long-period phase dual-period layered structure by compounding a specific composition and ratio of ceramide with rare ginsenoside CK, and then combining it with a temperature-controlled emulsification process. These nanolipid aggregates can achieve a dual enhancement of skin barrier repair and anti-aging effects, while efficiently meeting the needs of comprehensive barrier repair and anti-aging. Therefore, the nanolipid aggregates of this invention have promising application prospects. Attached Figure Description

[0012] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1 The results show the relative expression levels of Filaggrin mRNA in cells under different treatments in Experiment Example 5. Figure 2 The results show the expression levels of tight junction proteins in cells under different treatments in Experiment Example 5. Figure 3 The results show the secretion of type I procollagen in cells under different treatments in Experiment Example 5; Figure 4 The results show the expression levels of MMP-1 in cells under different treatments in Experiment Example 5. Detailed Implementation

[0014] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0015] To repair the skin barrier, current skincare products often contain ceramides. While these formulations provide basic barrier repair, the added ceramides are often single subtypes (such as NP) or mixtures with unclear proportions, lacking a systematic reconstruction of the stratum corneum's lipid layer structure. Research indicates that a complete skin barrier requires both short-cycle layer structures (approximately 6 nm, dominated by short-chain ceramides such as NP / AP, also known as the short-cycle phase, SPP) and long-cycle layer structures (approximately 13 nm, connected to adjacent lipid layers by long-chain ceramides such as EOS / EOP, also known as the long-cycle phase, LPP). Existing formulations typically contain very low levels or no EOS / EOP. Even when sufficient EOS / EOP ceramides are present, mismatched manufacturing processes result in insufficient formation or a low proportion of long-cycle layer structures, thus limiting the integrity and durability of barrier repair.

[0016] Furthermore, to simultaneously achieve skin barrier repair and anti-aging, those skilled in the art often combine ceramides with the anti-aging active ingredient ginsenosides. However, there is a contradiction between the solubility of ceramides and the thermal stability of ginsenosides. Ceramides have a high melting point (NP approximately 100~110℃), requiring high-temperature dissolution; glycosidic bonds may hydrolyze and become inactive at temperatures above 50℃. To address this, ceramides are typically added at room temperature in the final stage, but at low temperatures, ceramides easily crystallize, resulting in uneven product and reduced efficacy. To address these issues, Chinese patent CN1211073C uses an injection method to inject molten ceramide into an aqueous solution of ginsenosides to prepare a ginsenoside-ceramide liposome emulsion. However, due to the unclear composition of ceramides, precise biomimetic hierarchical assembly cannot be achieved; moreover, this preparation method passively encapsulates ginsenosides, resulting in low encapsulation rates, larger particle sizes, and a lower LPP content.

[0017] Based on the above problems, this invention combines a specific type of ceramide with a specific composition and ratio, and further optimizes the preparation method to obtain a biomimetic dual-period layered structure of lipid nanoparticles. This lipid nanoparticle has a biomimetic dual-period layered structure of short-period phase + long-period phase, which is formed by embedding rare ginsenoside CK in a lipid bilayer. The molar proportion of the long-period phase in the lipid bilayer is >60%, which is highly consistent with the layered structure of natural human stratum corneum lipids, and can provide a structural basis for the integrity and durability of barrier repair.

[0018] In this invention, the lipid bilayer is formed by the self-assembly of a composite ceramide and lipid components. The applicant has already filed a patent application for this composite ceramide, application number 2026109827652.

[0019] The aforementioned composite ceramide comprises four ceramide subtypes (NP, AP, EOP, and EOS), as well as phytosterols, behenol, and hydrogenated lecithin. The mass ratio of NP:AP:EOP:EOS is 10:2.5:1:1. In this composite ceramide, NP and AP form SPP, constructing the basic framework of the lipid bilayer. The extremely long ω-hydroxy fatty acid chains (C28-C34) of EOS and EOP span the entire thickness of the bilayer, acting as molecular anchors to connect adjacent lipid layers, forming LPP. The sphingosine bases of EOS also participate in the covalent cross-linking of keratinocyte membrane proteins. This composite ceramide achieves significant structural enhancement even at low concentrations (0.3% each of EOS and EOP) (LPP molar percentage increases from 0% to 62%).

[0020] The total amount of the four ceramides was in a mass ratio of 3:1:1 to behenol and phytosterol. The above ratio was based on the lipid composition of human skin. The lipid bilayer prepared by the above ratio has a specific biomimetic layered structure.

[0021] Specifically, phytosterols regulate membrane fluidity; behenol serves as the lipid matrix framework; and hydrogenated lecithin is used for bilayer formation.

[0022] The ginsenoside of this invention is a rare ginsenoside CK (CAS: 39262-14-1, molecular weight 622.88), which is a degradation product of ginsenoside Rb1 by β-glucosidase biotransformation. It contains only one glucose unit and its water solubility and transdermal absorption rate are significantly better than the original ginsenoside Rb1 (molecular weight 1109.26, tetrasaccharide).

[0023] Furthermore, the embodiments of this invention utilize rare ginsenoside CK, which was independently developed by the applicant in the early stages. The patent application number for this technology is 202411221196.7. Compared to commercially available rare ginsenoside CK, the rare ginsenoside CK used in this invention has higher purity and can precisely promote collagen production and has better anti-aging efficiency.

[0024] In some embodiments, the mass ratio of compound ceramide to rare ginsenoside CK is 0.5~5:0.01~1. More preferably, the mass ratio of compound ceramide to rare ginsenoside CK is 1~3:0.05~0.5. More preferably, the mass ratio of compound ceramide to rare ginsenoside CK is 1~5:1. It has been verified that within the above ratio range, compound ceramide and rare ginsenoside CK can produce a significant synergistic effect. Further investigation revealed that the synergistic mechanism involves a dual-pathway positive feedback: NAEO repairs the lipid barrier (first line of defense), and CK upregulates ZO-1 / Claudin-1 tight junction protein (second line of defense). The two pathways mutually enhance each other, thereby producing a synergistic effect of barrier repair and anti-aging.

[0025] In some embodiments, the lipid component includes cholesterol, stearic acid, and squalane.

[0026] The present invention also provides a method for preparing the above-mentioned nanolipid aggregates, which includes: mixing composite ceramide with lipid components, stirring continuously at 95°C until completely dissolved and in a uniform and transparent state to obtain an oil phase; heating water to 55~70°C, adding a thickener and a pre-solution of rare ginsenoside CK, mixing well to prepare an aqueous phase; slowly adding the oil phase to the aqueous phase, and after homogenization and emulsification, completing the in-situ self-assembly of nanolipid aggregates.

[0027] In the above preparation method, the heating temperature for preparing the oil phase is set to 95℃ because it is higher than the actual dissolution temperature (80-90℃) of NP (100~110℃) in the butanediol / glycerol solvent system, and also higher than the melting point of phytosterol (>120℃, which is reduced to about 90℃ in this system), ensuring that all high melting point components are completely dissolved.

[0028] The heating temperature for preparing the aqueous phase is set to 55~70℃ because this temperature needs to be higher than the phospholipid phase transition temperature (about 50~55℃) to ensure that the phospholipid is in the liquid crystal phase during subsequent emulsification, while being much lower than the CK degradation temperature (>80℃).

[0029] In some embodiments, rare ginsenoside CK is prepared as a pre-solution by dissolving it in propylene glycol or butylene glycol (5 to 10 times the mass of CK). Pre-dissolving CK in polyol can improve its dispersion uniformity in the aqueous phase and avoid excessively high local concentrations.

[0030] In some embodiments, the homogenization speed is 3000~5000 rpm, and the emulsification time is 10~20 min.

[0031] At the instant the oil and water phases come into contact, a transient temperature band of 70-80°C is formed at the interface. This invention employs a temperature-controlled emulsification process with an oil phase of 95°C and an aqueous phase of 60-65°C. At the instant the oil and water phases come into contact, a transient thermal gradient window of 70-80°C is formed at the interface. This temperature is exactly at the optimal window for phospholipid self-assembly. The amphiphilic structure of CK allows it to spontaneously locate at the water-lipid interface of the lipid bilayer during this process, achieving in-situ co-assembly and embedding. This window simultaneously satisfies the time decoupling of three thermodynamic contradictions in a single emulsification step: (1) higher than the actual dissolution temperature of ceramide in the solvent system (80-90°C), ensuring complete dissolution; (2) higher than the phospholipid phase transition temperature (Tm approximately 50-55°C), ensuring that the phospholipids are in the liquid crystal phase and can spontaneously arrange themselves; (3) lower than the CK degradation temperature (>80°C glycosidic bond hydrolysis), ensuring bioactivity. This transient thermal gradient exists only at the instant of oil-water mixing and naturally disappears with emulsification, which is a dynamic process control method. In contrast to CN1211073C, which uses an isothermal injection method at 65°C+60°C and has no thermal gradient design, the preparation process of CN1211073C shows that the particle size, encapsulation efficiency, and LPP content of the milky white liposome emulsion obtained by CN1211073C decrease significantly after deviating from the thermal gradient design.

[0032] The rare ginsenoside CK selected in this invention, prepared using the aforementioned process, is an amphiphilic molecule (hydrophobic steroidal core + hydrophilic monoglucose) capable of actively participating in the self-assembly process of the lipid bilayer. Its active participation in self-assembly is manifested in the fact that rare ginsenoside CK is positioned at the water-lipid interface of the nano-aggregate lipid bilayer. Its molecular structure allows it to regulate the interfacial curvature and stability of nanoparticles, making it a co-assembly component of the nanostructure rather than a passive load. To further verify the active co-assembly of rare ginsenoside CK, this invention detected the encapsulation efficiency and particle size of the nano-lipid aggregates. The results showed that the encapsulation efficiency of rare ginsenoside CK reached 85.8% ± 2.5% (far exceeding the level achievable by passive diffusion); in the control experiment without NAEO, the CK encapsulation efficiency was <5%, ruling out the possibility of simple dissolution; the larger molecular weight tetrasaccharide prototype Rb1 had an encapsulation efficiency of only 42%~46% under the same conditions, and its particle size increased to 145~152 nm, proving that the monosaccharide structure of rare ginsenoside CK is a necessary condition for its participation in co-assembly.

[0033] The preparation method provided by this invention enables the self-assembly of a nanolipid aggregate with a precisely controlled biomimetic layered structure. This nanolipid aggregate possesses a biomimetic SPP+LPP dual-periodic layered structure and also boasts advantages such as smaller and more uniform particle size, higher encapsulation efficiency, and a significantly increased LPP content. This nanolipid aggregate can be applied in the preparation of skincare products.

[0034] Therefore, the present invention can also provide a skin care product comprising the above-mentioned nanolipid aggregates; by mass percentage, the skin care product comprises the following raw materials: 0.5%~5% water-soluble complex ceramide, 0.01%~1% rare ginsenoside CK, 0.5%~5% cholesterol, 0.5%~3% stearic acid, 1%~5% squalane, and 0.1%~0.5% thickener.

[0035] In some embodiments, the thickener may be carbomer, specifically carbomer 940, carbomer 980, or other types with thickening effects.

[0036] In some embodiments, the skin care product also includes moisturizers and preservatives.

[0037] Specific moisturizers may include glycerin, sodium hyaluronate, or mineral oil, olive oil, etc.; preservatives may include methylparaben, ethylparaben, propylparaben, butylparaben, ethylhexylglycerin, or phenoxyethanol. Those skilled in the art can select conventional moisturizers and preservatives as needed.

[0038] In some embodiments, the humectant is glycerin and the preservative is phenoxyethanol.

[0039] In some embodiments, the skincare product comprises, by weight percentage, the following ingredients: 1%~3% complex ceramides, 0.05%~0.5% rare ginsenoside CK, 0.5%~5% cholesterol, 0.5%~3% stearic acid, 1%~5% squalane, 0.1%~0.5% carbomer, 1%~10% glycerin, and 0.3%~1% phenoxyethanol.

[0040] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0041] Example 1 This embodiment provides a skincare product containing nanolipid aggregates, the raw materials of which are shown in Table 1: Table 1 Formulation Information

[0042] The preparation method of the above skin care products is as follows: (1) Oil phase preparation: Ceramide NAEO raw material, cholesterol, stearic acid, squalane, polyglycerol-3 diisostearate and vitamin E are mixed according to the addition ratio, heated in a 95°C water bath and stirred until completely dissolved into a uniform transparent liquid.

[0043] (2) Preparation of aqueous phase: Heat deionized water to 60~65℃, add glycerol and pre-dispersed carbomer aqueous solution. Dissolve CK powder in propylene glycol, add to the aqueous phase and stir evenly.

[0044] (3) One-step temperature-controlled emulsification (in-situ co-assembly): The 95℃ oil phase is slowly added to the 60~65℃ aqueous phase containing CK, and the mixture is homogenized and emulsified at 4000 rpm for 15 min to complete the in-situ self-assembly of biomimetic nanolipid aggregates.

[0045] (4) Cooling and solidification: Naturally cool to room temperature, the lipid layer of nano-aggregates changes from liquid crystal phase to gel phase, and the co-assembled CK is locked.

[0046] (5) Post-processing: Add sodium hyaluronate, panthenol, and phenoxyethanol, adjust the pH to 5.5-6.0 with triethanolamine, homogenize at low speed for 5 minutes to obtain the face cream product.

[0047] Example 2 This embodiment provides a skincare product containing nanolipid aggregates, the raw materials of which are shown in Table 2: Table 2 Formulation Information

[0048] The composition of the above-mentioned ceramide NAEO raw material is the same as in Example 1. The preparation method of this skincare product is as follows: (1) NAEO preheating: Take ceramide NAEO raw material, heat it in a 95℃ water bath, and stir until it is completely clear and transparent.

[0049] (2) Preparation of aqueous phase: Deionized water was heated to 60-65℃, and glycerol, betaine and nicotinamide were added in sequence and stirred to dissolve. CK was dissolved in butanediol and then added to the aqueous phase.

[0050] (3) One-step in-situ co-assembly: The preheated NAEO raw material is slowly added to the aqueous phase containing CK at 60-65℃, and homogenized and emulsified at 3000 rpm for 10 min to complete the in-situ self-assembly of biomimetic nanolipid aggregates.

[0051] (4) Cooling and curing: Cool naturally to room temperature.

[0052] (5) Post-processing: Add sodium hyaluronate, panthenol, and phenoxyethanol, stir evenly, adjust the pH to 5.5~6.0, and obtain the essence product.

[0053] The characterization data of the nanolipid aggregates prepared in Examples 1 and 2 were compared with those of the control group, as detailed below: (1) Z-average particle size and PDI detection Instrument: Malvern Zetasizer Nano ZS Method: Dynamic Light Scattering (DLS) Sample preparation: Dilute the nanolipid aggregate dispersion with deionized water to an appropriate concentration (e.g., 0.1%), sonicate for 2 min to remove air bubbles, and measure at 25℃.

[0054] Parameters: scattering angle 173°, laser wavelength 633nm, each sample was measured 3 times and the average value was taken.

[0055] (2) Detection of Zeta potential Instrument: Malvern Zetasizer Nano ZS Method: Electrophoretic light scattering method Sample preparation: Same as above Parameters: Temperature 25℃, each sample was measured 3 times.

[0056] (3) CK encapsulation rate test Methods: Ultrafiltration centrifugation combined with HPLC Specific steps: Take the nanolipid aggregate dispersion, add it to an ultrafiltration centrifuge tube (molecular weight cutoff 10kDa), centrifuge at 4000rpm for 20min, and collect the filtrate; determine the free CK content in the filtrate by HPLC; encapsulation efficiency = (total CK amount - free CK amount) / total CK amount × 100%.

[0057] HPLC conditions: C18 column, mobile phase acetonitrile-water (gradient elution), detection wavelength 203 nm. Table 3 shows the characterization results: Table 3 Characterization results

[0058] A formulation containing only an equal amount of CK (CK dissolved alone in the aqueous phase without self-assembly) without the complex ceramide was used as a control. DLS analysis showed that the control group had no obvious nanoparticle peaks, and the CK encapsulation rate was <5%. This result indicates that the nanostructure is formed by the self-assembly of ceramide / phospholipid / poloxam in the complex ceramide, rather than by the simple dissolution of CK itself.

[0059] Experimental Example 1 This experiment compared the effects of nanolipid aggregates prepared under different types and ratios of ceramides. The specific experimental design is shown in Table 4. Table 4 Experimental Design

[0060] Nanolipid aggregates were obtained according to the preparation method of Example 1, and were characterized (as above) and compared with transdermal permeation in a Franz diffusion cell.

[0061] The Franz diffusion cell percutaneous permeation detection method is as follows: A Franz vertical diffusion cell (diffusion area 1.77 cm²) was used. 2A 12 mL receiving pool was used to fix the abdominal skin of an isolated SD rat between the supply and receiving pools, with the dermis facing down. The receiving solution was pH 7.4 PBS (containing 20% ​​ethanol), circulated at 37℃ with stirring at 600 rpm. 100 mg of sample was evenly applied to the skin surface and blocked with Parafilm. 0.5 mL samples were taken at 0.5, 1, 2, 4, 6, 8, 12, and 24 h, and an equal volume of receiving solution was added immediately. After 24 h, the skin was removed, the surface residue was rinsed with physiological saline, and the stratum corneum was collected using the adhesive tape peeling method (15 times). The remaining skin was minced and extracted with methanol / chloroform (1:1) by ultrasonication. Ceramides were detected by HPLC-ELSD, and ginsenoside CK was detected by HPLC-UV (203 nm). The cumulative transdermal release, steady-state transdermal flux, and skin retention were calculated. Each group had n=6.

[0062] The characterization results of the nanocarriers are shown in Table 5: Table 5 Comparison of Nanocarrier Characterization Results

[0063] The SAXS layered structure characterization results are shown in Table 6: Table 6 Characterization results of SAXS layered structures

[0064] The results of percutaneous perfusion in the Franz diffusion cell are shown in Table 7: Table 7 Results of transdermal penetration

[0065] The results in Tables 5 and 6 show that the nanolipid aggregates prepared by the composite ceramide of the present invention have smaller, more uniform, and more stable particle sizes, and the long-period phase accounts for the highest proportion of the formed nanolipid aggregates, reaching over 60%. Table 7 shows that the nanolipid aggregates prepared by the composite ceramide of the present invention have the best transdermal penetration effect.

[0066] Experiment Example 2 This experiment compares the skin barrier function of compound ceramides and rare ginsenoside CK at different ratios. The specific experimental design is shown in Table 8. Table 8 Experimental Design

[0067] Filaggrin is a key structural protein of the stratum corneum, and its upregulation directly reflects enhanced barrier differentiation function. The expression level of filaggrin mRNA was detected by RT-qPCR: HaCaT cells were expressed at a concentration of 2 × 10⁻⁶ mRNA. 5Filagrin mRNA was inoculated into 6-well plates and cultured for 24 h. After adhesion, the cells were treated with the appropriate concentrations for 48 h. Total RNA was extracted using the TRIzol method, and cDNA was synthesized via reverse transcription. GAPDH was used as an internal control. The relative expression level of Filaggrin mRNA was detected by SYBR Green quantitative PCR, and ploidy change was calculated using the 2^(-ΔΔCt) method.

[0068] The synergistic effect of each group is then calculated using the synergistic ratio (CER) value, as follows: Calculation of the summation of expected values: FC_expected = FC_NAEO + FC_CK-1 (where FC is the ploidy change of each group relative to the blank control).

[0069] Synergistic effect ratio: CER = FC_observed / FC_expected.

[0070] Interpretation criteria: CER>1.1 indicates synergistic effect (the measured effect significantly exceeds the sum of expectations), 0.9≤CER≤1.1 is an additive effect, and CER<0.9 is an antagonistic effect.

[0071] This method directly compares the measured effect with the theoretical summation expectation, with clear mathematical meaning. It does not require additional dose-response curve fitting and is suitable for screening experiments with multiple ratios at single concentration levels.

[0072] The test results are shown in Table 9: Table 9. Filaggrin expression and CER values ​​in each group

[0073] The above results indicate that within the NAEO:CK ratio range of 1:1 to 5:1, the CER remains stable at 1.38-1.43, with a synergistic effect of 38%-43%, demonstrating significant synergy. When the ratio is <1:1 (CK excess, G1-G2), the CER is <0.9, showing antagonism; when the ratio is >5:1 (NAEO excess, G8-G9), the CER is ≤1.1, and the synergy disappears. Therefore, 1:1 to 5:1 represents the narrow ratio window with the strongest synergistic effect.

[0074] Experimental Example 3 This experiment compared the characterization results of nanolipid aggregates prepared under different ginsenoside addition conditions. The specific experimental design is shown in Table 10. Table 10 Experimental Design

[0075] The above design was used to prepare nanolipid aggregates according to the preparation method of Example 1, and the results are shown in Table 11: Table 11 Characterization Results

[0076] Groups T4, T5, and T6 could not form nanolipid aggregates. The results of groups T1, T2, and T3 showed that, compared with equal mass or equimolar ratio of Rb1, the nanolipid aggregates prepared with rare ginsenoside CK in group T1 had higher encapsulation efficiency and smaller particle size.

[0077] Experiment Example 4 This experiment aims to compare the preparation of nanolipid aggregates under different heating temperatures. The specific experimental design is shown in Table 12. Table 12 Experimental Design

[0078] Nanolipid aggregates were obtained according to the preparation method of Example 1, and they were characterized and compared with transdermal permeation in a Franz diffusion cell.

[0079] The characterization results of the nanocarriers are shown in Table 13: Table 13 Comparison of Nanocarrier Characterization Results

[0080] The SAXS layered structure characterization results are shown in Table 14: Table 14 Characterization results of SAXS layered structures

[0081] The results of percutaneous perfusion in the Franz diffusion cell are shown in Table 5: Table 15 Results of transdermal penetration

[0082] The results in Tables 13 and 14 show that the nanolipid aggregates prepared by the composite ceramide of the present invention have smaller, more uniform, and more stable particle sizes, higher encapsulation efficiency, and the highest proportion of long-cycle phases in the formed nanolipid aggregates, reaching over 60%. Table 15 shows that the nanolipid aggregates prepared by the composite ceramide of the present invention have the best transdermal penetration effect.

[0083] Experimental Example 5 This experiment verified the effects of different concentrations of compound ceramides and / or rare ginsenosides (CK) using in vitro cell experiments, as detailed below: Cell models: HaCaT keratinocytes (barrier repair function) / HDFa skin fibroblasts (anti-aging function). n=6 per group.

[0084] 1. Cytotoxicity assay The cell viability at different concentrations is shown in Table 16: Table 16 Cell viability at different concentrations

[0085] The experimental concentrations were determined to be 0.5% (v / v, the dilution ratio of NAEO raw material in the culture medium) and 0.5% (w / v, the concentration of CK in the culture medium). The survival rates of both groups were >90% at 24h and 48h.

[0086] 2. Filaggrin mRNA expression (barrier repair function) Experimental method: HaCaT cells were used at a concentration of 2 × 10⁻⁶. 5 Filagrin mRNA was inoculated into 6-well plates and cultured for 24 h. After adhesion, the cells were treated with the appropriate concentrations for 48 h. Total RNA was extracted using the TRIzol method, and cDNA was synthesized via reverse transcription. GAPDH was used as an internal control. The relative expression level of Filaggrin mRNA was detected by SYBR Green quantitative PCR, and ploidy change was calculated using the 2^(-ΔΔCt) method.

[0087] In this experiment, the ratio of NAEO to CK was 1:1. This ratio was chosen because, in the previous gradient experiments, the synergistic effect of the NAEO:CK = 1:1 ratio group was second only to the 2:1 group, approximately 93% of that of the 2:1 group, but the detection cost was reduced by 30%. Therefore, this ratio was adopted in the effect verification.

[0088] The test results are shown in Table 17 and Figure 1 As shown: Table 17. Relative expression levels of Filaggrin mRNA in cells under different treatments

[0089] From Table 17 and Figure 1 The results showed that the expression level in the combination group was about 2.8 times that of the control group, which was significantly higher than the simple sum of NAEO alone (about 1.6 times) and CK alone (about 1.4 times), suggesting synergistic effect.

[0090] 3. Claudin-1 protein expression (barrier repair function) Experimental methods: HaCaT cells were treated in groups for 48 hours, and lysates were collected. Protein quantification was performed using the BCA method. Equal amounts of protein were separated by 10% SDS-PAGE electrophoresis, transferred to a PVDF membrane, blocked with 5% skim milk powder for 1 hour, and incubated overnight at 4°C with ZO-1 and Claudin-1 primary antibodies, respectively. The membrane was then incubated with HRP-labeled secondary antibody at room temperature for 1 hour. ECL chemiluminescence imaging was performed, with β-actin as an internal control. Gray-scale values ​​were quantified using ImageJ software. The detection results are shown in Table 18 and... Figure 2 As shown: Table 18 Expression levels of tight junction proteins in cells under different treatments

[0091] Claudin-1 is a key transmembrane protein for tight junctions. The expression level in the combined group was approximately 2.8 times that of the control group, exceeding the sum of the individual effects of the two groups, demonstrating that the combined group had a synergistic effect.

[0092] 4. Procollagen I secretion (anti-aging effect) Experimental method: HDFa cells were prepared at a concentration of 1×10⁻⁶. 5 Cells were seeded into 24-well plates and cultured for 24 hours. After adhesion, the cells were treated with the appropriate concentration of the drug for 48 hours, and the supernatant was collected. The human Procollagen I ELISA kit was used according to the manufacturer's instructions: 100 μL of sample was added per well, incubated at 37°C for 2 hours, washed 5 times, the detection antibody was added for 1 hour, TMB substrate was added for 15 minutes of color development, and the reaction was terminated with stop solution. The OD value was read at 450 nm using a microplate reader, and the secretion volume (ng / mL) was calculated based on the standard curve.

[0093] The test results are shown in Table 19 and Figure 3 As shown: Table 19. Type I procollagen secretion under different treatments

[0094] Procollagen I is a direct indicator of collagen synthesis. The secretion of the compound group was about 2.8 times that of the control group, far exceeding that of NAEO alone (about 1.4 times) and CK alone (about 1.7 times), proving that the compound group has a synergistic effect.

[0095] 5. Matrix metalloproteinase-1 (MMP-1) expression (anti-aging effect) Experimental Methods: HDFa cells were treated in groups for 48 hours, and the culture supernatant was collected. A human MMP-1 ELISA kit was used, with 100 μL / well added and incubated at 37℃ for 2 hours. After washing 5 times, biotinylated antibody was added and incubated at room temperature for 1 hour. HRP-streptavidin was added for 30 minutes, TMB color development was performed for 15 minutes, and the assay was terminated with stop solution. OD values ​​were read at 450 nm, and MMP-1 expression (ng / mL) was quantified using the standard curve method. Lower MMP-1 values ​​indicate better collagen protection. The results are shown in Table 20 and... Figure 4 As shown: Table 20. Expression levels of MMP-1 in cells under different treatments

[0096] MMP-1 is a key enzyme that degrades collagen, and lower expression levels indicate better collagen protection. In the combination group, MMP-1 levels decreased to approximately 29% of the control group, demonstrating a significantly better inhibitory effect than any of the single-agent groups, proving that the combination group has a synergistic effect.

[0097] Experimental Example 6 This experiment conducted human efficacy testing. The matrix of the four test products was completely consistent with the formulation of Example 1, with only the core active ingredients differing (blank matrix / NAEO 1.0% / CK 0.2% / NAEO 1.0%+CK 0.2%). The specific tests are as follows: 1. TEWL Test (Barrier Repair Efficacy): Five healthy volunteers (aged 18-45) were selected, and the TEWL test was performed on the inner forearm. The test time was 8 hours after product use. The test results are shown in Table 21: Table 21 TEWL Test Results

[0098] The results showed that the NAEO+CK group had the lowest transdermal water loss compared to the inactive ingredient, single NAEO, or single CK groups.

[0099] 2. Wrinkle Depth Test (Anti-aging Efficacy): Five healthy volunteers (aged 18-45) were selected, and the depth of wrinkles around the eyes was measured. The treatment was administered continuously for 8 weeks. The test results are shown in Table 22. Table 22 Wrinkle Depth Test Results

[0100] The results showed that, compared with the groups without active ingredients, single NAEO, or single CK, the wrinkle depth in the NAEO+CK group was significantly reduced.

[0101] Experimental Example 7 This experiment verified the stability of the face cream prepared in Example 1. It was placed in ovens at room temperature, 5°C, and 45°C for specific times, and the results are shown in Table 23. Table 23 Stability Results

[0102] As can be seen from the results in Table 23, the face cream containing nanolipid aggregates prepared in this invention has good stability.

[0103] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A nanolipid aggregate with a biomimetic dual-periodic layered structure, characterized in that, The nanolipid aggregates have a biomimetic short-period phase + long-period phase double-period layered structure, which is formed by embedding rare ginsenoside CK in a lipid bilayer. The long-period phase has a molar percentage of >60% in the lipid bilayer; the lipid bilayer is composed of a complex ceramide and lipid components; the complex ceramide includes ceramide subtypes NP, AP, EOP, and EOS, with a mass ratio of 10:2.5:1:

1. The mass ratio of the compound ceramide to the rare ginsenoside CK is 0.5~5:0.01~1.

2. The nanolipid aggregate according to claim 1, characterized in that, The composite ceramide also includes phytosterols, behenol, and hydrogenated lecithin; The total mass ratio of the ceramide subtypes NP, AP, EOP, and EOS to behenol and phytosterol is 3:1:1:

1.

3. The nanolipid aggregate according to claim 2, characterized in that, The lipid components include cholesterol, stearic acid, and squalane; The mass ratio of the composite ceramide to cholesterol, stearic acid and squalane is 0.5~5:0.5~5:0.5~3:1~5.

4. The nanolipid aggregate according to claim 1, characterized in that, The mass ratio of the compound ceramide to the rare ginsenoside CK is 1~3:0.05~0.

5.

5. The nanolipid aggregate according to claim 4, characterized in that, The mass ratio of the composite ceramide to the rare ginsenoside CK is 1~5:

1.

6. The method for preparing nanolipid aggregates according to any one of claims 1 to 5, characterized in that, include: The composite ceramide was mixed with the lipid component and stirred continuously at 95°C until completely dissolved and in a uniform and transparent state to obtain the oil phase; Heat water to 55-70°C, add thickener and the pre-solution of the rare ginsenoside CK, mix well to form an aqueous phase; The oil phase is slowly added to the aqueous phase, and after homogenization and emulsification, the in-situ self-assembly of the nanolipid aggregates is completed.

7. The preparation method according to claim 6, characterized in that, The homogenization speed is 3000~5000 rpm, and the emulsification time is 10~20 min.

8. The application of the nanolipid aggregates as described in any one of claims 1 to 5 or the nanolipid aggregates prepared by the preparation method described in claim 6 or 7 in the preparation of skin care products.

9. A skincare product, characterized in that, It includes the nanolipid aggregates according to any one of claims 1 to 5; The skincare product comprises the following ingredients by weight percentage: 0.5%~5% of the compound ceramide, 0.01%~1% of rare ginsenoside CK, 0.5%~5% of cholesterol, 0.5%~3% of stearic acid, 1%~5% of squalane, and 0.1%~0.5% of thickener.

10. The skincare product according to claim 9, characterized in that, The skincare products also include moisturizers and preservatives; The skincare product comprises the following ingredients by weight percentage: 1%~3% of the compound ceramide, 0.05%~0.5% of rare ginsenoside CK, 0.5%~5% of cholesterol, 0.5%~3% of stearic acid, 1%~5% of squalane, 0.1%~0.5% of thickener, 1%~10% of moisturizer, and 0.3%~1% of preservative.

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