Preparation method and application of ginsenoside-loaded triple-assembled pickering emulsion

Abstract

The application relates to a preparation method and application of a ginsenoside-loaded triple-assembled Pickering emulsion, and belongs to the biomedical technology field. In order to solve the problems that the Pickering emulsion constructed by natural active ingredients in the prior art has insufficient mechanical strength of an interface film, is easy to break and lose stability, has passive release behavior, lacks controllability and environmental responsiveness, and is difficult to realize precise release, a ginsenoside-loaded triple-assembled Pickering emulsion is provided. Through a three-step cascade strategy of "molecule-interface-function", a stable "metal-phenol coordination network" interface layer is constructed on the surface of oil droplets, the physical and chemical stability of the emulsion is fundamentally improved, a Pickering emulsion which is all-natural, highly stable and has a pH response intelligent release function is successfully constructed, and the triple-assembled Pickering emulsion has significant advantages in stability, controllable release and function integration, and provides a feasible technical strategy for a response type delivery system design based on natural products.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a method for preparing and applying a triple-assembled Pickering emulsion loaded with ginsenosides. Background Technology

[0002] Pickering emulsions, a unique emulsion system where solid particles adsorb at the oil-water interface and play a major stabilizing role, are fundamentally different from traditional emulsions that rely on small-molecule surfactants. Their core advantage lies in the mechanical barrier formed by the solid particles at the interface, which more effectively resists aggregation, Ostwald ripening, and phase separation, thus providing superior long-term physical stability. Simultaneously, it avoids the skin irritation, biotoxicity, and environmental residue problems that may arise from small-molecule surfactants, resulting in higher safety. Furthermore, the particle interface itself can serve as a designable and functionalizable platform, endowing the emulsion with intelligent properties such as responsiveness and targeting through chemical modification or physical assembly, opening new avenues for developing advanced delivery systems. Therefore, Pickering emulsions have significant application prospects in fields with high safety and functional requirements, such as food, cosmetics, and transdermal drug delivery.

[0003] Currently, Pickering emulsions constructed from natural active ingredients such as proteins and polysaccharides have become a research hotspot, aiming to pursue higher safety and environmental friendliness. However, they still face two common bottlenecks in practical applications: First, the interfacial film formed by natural particles has limited mechanical strength and is prone to damage under long-term storage or external force, leading to emulsion instability. Second, its release behavior is mostly passive diffusion, lacking controllability and environmental responsiveness, making it difficult to achieve precise release of active ingredients at specific times and locations. This seriously restricts its value in functional skin care products and drug delivery systems. Summary of the Invention

[0004] This invention addresses the problems of insufficient interfacial membrane mechanical strength, easy breakage and instability, and passive release behavior in Pickering emulsions constructed from natural active ingredients in the prior art, which lack controllability and environmental responsiveness, making it difficult to achieve precise release. It provides a method for preparing and applying a triple-assembled Pickering emulsion loaded with ginsenosides.

[0005] One objective of this invention is to provide a method for preparing a ginsenoside-loaded triple-assembled Pickering emulsion, the preparation method comprising the following steps: S1: Weigh out ginsenosides and dissolve them in anhydrous ethanol, then heat to aid dissolution, to obtain solution A; weigh out grape seed extract and dissolve it in deionized water preheated to 45±2℃, to obtain solution B; under magnetic stirring at 400 rpm, add solution A dropwise to solution B, then place the beaker in a 45℃ constant temperature water bath and continue stirring for 30-40 min, then cool to room temperature to obtain a binary self-assembled suspension; S2: Weigh squalane and tocopherol into a beaker, place the beaker in a 40°C water bath, and stir until the mixture is completely homogeneous and transparent to obtain the oil phase; S3: The binary self-assembled suspension prepared in S1 was used as the aqueous phase and transferred to the processing cup of a high-speed shear emulsifier for emulsification at 12,000 rpm. Under high-speed shear conditions, the preheated oil phase prepared in S2 was added dropwise to the aqueous phase at a uniform rate for 5 min. After the addition was completed, high-speed shear emulsification was continued at 12,000 rpm for 15 min to obtain the primary pre-emulsion. S4: Weigh zinc gluconate and dissolve it in deionized water to obtain a zinc ion solution; under continuous stirring at 500 rpm, add the zinc ion solution dropwise to the primary pre-emulsion obtained in S3 for 5 min; after the addition is complete, continue stirring for 30 min to obtain the intermediate pre-emulsion. S5: The intermediate pre-emulsion prepared in S4 was transferred to a high-pressure homogenizer and homogenized for 3 cycles at a pressure of 60 MPa. After homogenization, the pH was adjusted with 0.1 mol / L sodium hydroxide solution, and then the preservative ketone was added. Deionized water was added to obtain an emulsion mixture. The emulsion mixture was stirred at low speed at 200 rpm for 60 min at room temperature to obtain a triple-assembled Pickering emulsion.

[0006] In a preferred embodiment of the present invention, the mass-to-volume ratio of ginsenosides and anhydrous ethanol in S1 is 100 mg: 2 mL.

[0007] In a preferred embodiment of the present invention, the mixed mass-volume ratio of grape seed extract and deionized water in S1 is 100 mg: 68 mL.

[0008] In a preferred embodiment of the present invention, the mass ratio of squalane and tocopherol in S2 is 8:0.5.

[0009] In a preferred embodiment of the present invention, the mixing mass ratio of zinc gluconate and deionized water in S4 is 0.5:10.

[0010] In a preferred embodiment of the present invention, the pH value in S5 is adjusted to 7.0 ± 0.1.

[0011] In a preferred embodiment of the present invention, the amount of preservative ketone added in S5 is 0.1% of the total mass of Pickering emulsion.

[0012] The second objective of this invention is to provide a ginsenoside-loaded triple-assembled Pickering emulsion, which is obtained by the above-described preparation method.

[0013] A third objective of this invention is to provide the application of the above-mentioned triple-assembled Pickering emulsion in the preparation of anti-aging cosmetics.

[0014] The fourth objective of this invention is to provide the application of the above-mentioned triple-assembled Pickering emulsion in the preparation of transdermal drug delivery formulations.

[0015] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention successfully constructs a ginsenoside-loaded triple-assembled pickering emulsion. Based on the self-assembly of ginsenosides and grape seed extract and in-situ cross-linking at the zinc ion interface, this novel pickering emulsion utilizes a three-step cascade strategy of "molecule-interface-function" to construct a stable "metal-phenol coordination network" interface layer on the oil droplet surface, fundamentally improving the physicochemical stability of the emulsion. Compared with existing natural pickering emulsions, the triple-assembled pickering emulsion provided by this invention achieves significant improvements and breakthroughs in dosage form design, release mechanism, and efficacy system.

[0016] Firstly, regarding dosage form construction and long-term stability: This invention constructs a robust interfacial barrier through a step-by-step assembly of "molecule-nanoparticle-interfacial cross-linking network". Firstly, utilizing the amphiphilic glycoside structure of ginsenosides and the hydrophobic interactions and hydrogen bonds between grape seed extract polyphenol molecules, well-structured nanocomposite particles are self-assembled in the aqueous phase – this is the basic step. Subsequently, during emulsification, these nanoparticles efficiently adsorb onto the newly formed oil-water interface. The most crucial step is the introduction of biocompatible zinc ions (zinc gluconate), which undergo in-situ coordination cross-linking with the abundant phenolic hydroxyl groups of grape seed extract at the interface, forming a robust "metal-phenol coordination network" on the surface of each oil droplet. This chemically cross-linked layer greatly enhances the mechanical strength and integrity of the interfacial film, effectively resisting interfacial deformation and damage, thus solving the fundamental problem of fragile interfacial films and insufficient long-term stability in traditional natural particulate emulsions.

[0017] Secondly, regarding the controllability of the release behavior: This invention achieves regulation of the release process based on the pH response characteristics of metal-phenol coordination bonds. Under near-neutral conditions, the coordination network structure is stable and can effectively maintain the encapsulated state of the contents; when the system is in a weakly acidic environment, the intervention of hydrogen ions leads to the dissociation of coordination bonds, and the interface structure undergoes controllable loosening, thereby triggering the directional release of active substances; this mechanism enables the emulsion to respond sensitively to the weakly acidic environment of the skin surface, realizing the on-demand release of active ingredients at the application site, overcoming the limitations of the blind and uncontrollable release process of traditional Pickering emulsions.

[0018] Third, regarding multifunctional synergistic integration: This invention constructs a multifunctional, integrated system. Firstly, its core active ingredient, ginsenoside, has been widely proven to possess significant anti-inflammatory, skin barrier repair-promoting, and photoaging-delaying bioactivities, endowing the emulsion with potential therapeutic application value. In addition to its basic function as a carrier, grape seed extract and tocopherol in the system synergistically exert antioxidant effects, effectively addressing oxidative stress; simultaneously, the oil phase squalane strengthens the skin barrier and provides long-lasting hydration. This system not only achieves precise delivery of active ingredients but also, through the functional integration of carrier and contents, exerts synergistic effects in skin repair, anti-oxidation, and hydration, forming a "multifunctional" delivery platform, providing a feasible path for developing multifunctional, efficacy-oriented products.

[0019] Fourth, it is safe, efficient, and has broad application prospects: the all-natural ingredients ensure good biocompatibility and low irritation, and transdermal penetration experiments have confirmed that it can effectively promote the transdermal absorption and skin retention of active ingredients. This research provides innovative solutions and experimental evidence for the development of environmentally responsive, highly efficient all-natural cosmetics and transdermal drug delivery formulations.

[0020] In summary, the core of this invention lies in a three-step cascade assembly strategy of "molecule-interface-function," the working principle of which is as follows: First, at the molecular level, the amphiphilic structural units of ginsenosides and polyphenolic components in grape seed extract self-assemble through hydrophobic interactions and hydrogen bonding networks to form uniformly sized nanocomposite particles rich in phenolic hydroxyl groups on their surface; these particles themselves possess both drug-carrying capacity and bioactivity. Second, during emulsification, based on the Pickering emulsification mechanism, these amphiphilic nanoparticles are irreversibly adsorbed onto the newly formed oil-water interface, forming a preliminary particle stabilization layer and realizing the localization of functional units on the interface. Most importantly, by introducing biocompatible zinc ions (such as zinc gluconate), they selectively undergo in-situ coordination crosslinking with the high density of phenolic hydroxyl groups on the surface of the interfacial particles, thereby constructing a robust "metal-phenol coordination network" with dynamic response characteristics on the oil droplet surface. This design achieves directional reinforcement of the interfacial region.

[0021] This cascade approach of "self-assembly positioning-in-situ cross-linking enhancement" not only improves the mechanical strength of the natural particulate interface membrane through chemical cross-linking, but also utilizes the pH sensitivity of the metal-phenol coordination bond to enable the emulsion to have environmentally responsive release capabilities. The entire system achieves structural integration and performance enhancement from active ingredients to functionalized carriers. Attached Figure Description

[0022] Figure 1 Particle size distribution of binary self-assembled ginsenoside-grape seed extract; Figure 2Zeta potential diagram of the binary self-assembly of ginsenoside-grape seed extract; Figure 3 Scanning electron micrograph of the binary self-assembly of ginsenoside-grape seed extract; Figure 4 Particle size distribution of Pickering emulsion 1; Figure 5 Zeta potential diagram of Pickering emulsion 1; Figure 6 Confocal microscopy image of Pickering emulsion 1; Figure 7 Fourier transform infrared spectrum of the zinc ion-grape seed extract coordination product; Figure 8 Storage stability plots for different treatment groups under different conditions; Figure 9 Storage stability graphs for different treatment groups under accelerated conditions at 55°C; Figure 10 Figures showing the emulsion state of different treatment groups after stability testing; Figure 11 Statistical chart of the retention rate of total ginsenosides in different treatment groups; Figure 12 The in vitro release curves of ginsenosides from different treatment groups in different pH media are shown; A is Pickering emulsion 1, B is control emulsion 1, C is control emulsion 2, and D is control emulsion 3. Figure 13 Zn was released from Pickering emulsion 1 under different pH treatments. 2+ Concentration statistics chart; Figure 14 Statistical graph of DPPH free radical scavenging rate for different treatment groups; Figure 15 ICs for different processing groups 50 Value comparison chart; Figure 16 Statistical graph showing the change in skin stratum corneum moisture content over time for different treatment groups; Figure 17 Statistical graph showing the change in skin hydration rate over time for different treatment groups; Figure 18 Figure 1 shows the cytotoxicity of HaCaT cells under different treatment groups. Figure 19 The steady-state shear flow curve of Pickering emulsion 1 is shown. Figure 20 The diagram shows the dynamic viscoelastic properties of the emulsions in different treatment groups; A is Pickering emulsion 1, B is control emulsion 1, C is control emulsion 2, and D is control emulsion 3. Figure 21 In vitro transdermal permeation curves of ginsenosides from different treatment groups; Figure 22 A comparison chart showing the retention levels of ginsenosides in the skin of different treatment groups. Detailed Implementation

[0023] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0025] Example 1: Preparation of binary self-assemblies of ginsenosides and grape seed extract 100 mg of ginsenosides were weighed and dissolved in 2 mL of anhydrous ethanol, and heated to aid dissolution, to obtain solution A; 100 mg of grape seed extract (purity ≥95%) was weighed and dissolved in 68 mL of deionized water preheated to 45±2℃, to obtain solution B; under magnetic stirring at 400 rpm, solution A was added dropwise to solution B, and then the beaker was placed in a constant temperature water bath at 45℃ and stirred for 30 min. After cooling to room temperature, a binary self-assembled suspension was obtained, which was sealed and stored at 4℃ for later use.

[0026] During this process, ginsenosides and grape seed extract spontaneously assemble into a nanoscale binary self-assembled suspension through intermolecular hydrophobic interactions and hydrogen bonds.

[0027] Results data: 1. Particle size and polydispersity index determination Take 1 mL of the binary self-assembled suspension prepared above and use a nanoparticle tracking analyzer to measure the diluted sample at 25℃. The instrument automatically analyzes and reports the average hydrodynamic particle size and polydispersity index of the particles. Each sample is measured in parallel 3 times and the average value is taken.

[0028] 2. Zeta potential measurement The zeta potential analysis module of the laser particle size analyzer was used for measurement. The above binary self-assembled suspension was diluted 100 times, and the diluted sample was injected into a special potential measurement cell. The instrument measured the zeta potential value of the particles by electrophoretic light scattering principle. Each sample was measured 3 times.

[0029] The results are as follows Figure 1-2 As shown, the average hydrodynamic diameter of the successfully prepared binary self-assembled body is 111.8 ± 8.3 nm. Figure 1 The polydispersity index was 0.17 ± 0.02. Figure 2 The value indicates that the particle size distribution is uniform; the Zeta potential value is -40.2±1.5 mV, indicating that the surface of the assembly carries a negative charge and the electrostatic repulsion between particles is strong, which is conducive to the formation of a stable colloidal dispersion system.

[0030] 3. Morphological observation using scanning electron microscopy To visually observe the microstructure and size of the aforementioned binary self-assembled structures, scanning electron microscopy was used for analysis. The binary self-assembled suspension was diluted 10-fold with ultrapure water, and 10 μL of the suspension was dropped onto a clean silicon wafer or conductive adhesive, allowing it to air dry at room temperature. After the sample was completely dry, a 5 nm thick gold-palladium alloy film was sputtered onto the sample surface using an ion sputtering apparatus to enhance its conductivity and prevent charge accumulation. The treated sample was then placed in the scanning electron microscope stage, and its surface morphology was observed and photographed at an accelerating voltage of 3.0 kV and a magnification of 80,000–100,000x.

[0031] The results are as follows Figure 3 As shown, the binary self-assembled particles are spherical or slightly ellipsoidal nanoparticles with good dispersibility, no obvious agglomeration, and clear particle edges.

[0032] Example 2: Preparation of a ginsenoside-loaded triple-assembled Pickering emulsion S1: Weigh 8.0 g of squalane and 0.5 g of tocopherol into a beaker, place the beaker in a 40°C water bath, and stir until the mixture is completely homogeneous and transparent to obtain the oil phase; S2: The binary self-assembled suspension prepared in Example 1 was used as the aqueous phase and transferred to the processing cup of a high-speed shear emulsifier for emulsification at 12,000 rpm. Under high-speed shear conditions, the preheated oil phase prepared in S1 was added dropwise to the aqueous phase at a uniform rate for 5 min. After the addition was completed, high-speed shear emulsification was continued at 12,000 rpm for 15 min to obtain a primary pre-emulsion. S3: Weigh 0.5 g of zinc gluconate and dissolve it in 10 g of deionized water to obtain a zinc ion solution; under continuous stirring at 500 rpm, add the zinc ion solution dropwise to the primary pre-emulsion obtained in S2 for 5 min; after the addition is complete, continue stirring for 30 min to ensure that the zinc ions fully migrate to the oil-water interface and complete the coordination cross-linking reaction with the grape seed extract to obtain the intermediate pre-emulsion; S4: Transfer the intermediate pre-emulsion prepared in S3 to a high-pressure homogenizer and homogenize it for 3 cycles at 60 MPa (to further reduce the droplet size and make the interface layer more uniform and dense). After homogenization, use 0.1 M sodium hydroxide solution to precisely adjust the pH of the emulsion system to 7.0 ± 0.1 with stirring. Then add 0.1 g of preservative ketone and add deionized water to a total mass of 100 g to obtain an emulsion mixture. Stir the emulsion mixture at low speed at 200 rpm for 60 min at room temperature to obtain Pickering emulsion 1, which is sealed and stored in a cool place at room temperature.

[0033] Comparative Example 1: This comparative example is used to investigate the key effects of zinc ion crosslinking on emulsion stability, rheological properties, and pH-responsive release behavior.

[0034] The difference between this comparative example and Example 2 is that the zinc gluconate solution was replaced with an equal mass of deionized water, while the other steps were the same as in Example 2, to obtain Comparative Emulsion 1.

[0035] Comparative Example 2: This comparative example is used to verify the necessity of grape seed extract as a phenolic hydroxyl ligand in the formation of interfacial cross-linked networks.

[0036] The difference between this comparative example and Example 2 is that the preparation steps of the binary self-assembled suspension in S2 are as follows: 100 mg of ginsenosides are weighed and dissolved in 2 mL of anhydrous ethanol, heated to aid dissolution, and 68 mL of deionized water preheated to 45±2℃ is added dropwise. The mixture is then stirred for 30 min in a constant temperature water bath at 45℃ and cooled to room temperature to obtain the binary self-assembled suspension, which is used as the aqueous phase. The other steps are the same as in Example 2 to obtain comparative emulsion 2.

[0037] Comparative Example 3: This comparative example is used to verify the key role of the "molecular-nanoparticle" level ordered self-assembly process in the formation of uniform and stable interfacial particles.

[0038] The difference between this comparative example and Example 2 is that the preparation steps of the binary self-assembled suspension in S2 are as follows: 100 mg of ginsenoside and 100 mg of grape seed extract are weighed and dissolved in 70 mL of room temperature deionized water, and the mixture is sheared at 10,000 rpm for 5 min using a high-speed disperser to obtain a suspension, which is used as the aqueous phase; the other steps are the same as in Example 2 to obtain comparative emulsion 3.

[0039] Comparative Example 4: This comparative example was used to evaluate the protective ability of different dosage forms on active ingredients. A traditional Tween 80 emulsion loaded with total ginsenosides was prepared. The oil phase composition was the same as that of Pickering emulsion 1 prepared in Example 2 (8.0 g squalane and 0.5 g tocopherol). The aqueous phase was a deionized water solution containing 2% (w / w) Tween 80 and an equal amount of total ginsenosides. The oil phase was slowly added to the aqueous phase, and high-speed shear emulsification was carried out at 12,000 rpm for 20 min. Then, it was homogenized under high pressure at 60 MPa for 3 cycles, and the pH was adjusted to 7.0 to obtain the traditional Pickering emulsion.

[0040] Comparative Example 5: To evaluate the biocompatibility of the carrier material itself, a conventional Tween 80 emulsion without active ingredients was prepared as a blank carrier. The oil phase composition was the same as that of Pickering emulsion 1 prepared in Example 2 (8.0 g squalane and 0.5 g tocopherol). The aqueous phase was a deionized aqueous solution containing only 2% (w / w) Tween 80, without any added active ingredients. The oil phase was slowly added to the aqueous phase, and high-speed shear emulsification was performed at 12,000 rpm for 20 min. Then, the mixture was homogenized under high pressure at 60 MPa for 3 cycles, and the pH was adjusted to 7.0 to obtain the blank Pickering emulsion.

[0041] Effect Experiment: 1. Characterization of basic physicochemical properties of emulsions (1) Measurement of droplet size, distribution and zeta potential Take 1 mL of Pickering emulsion 1 prepared in Example 2 and dilute it 100 times with deionized water to eliminate the multiple scattering effect. Use a laser particle size analyzer to determine the average droplet size, particle size distribution and zeta potential of the diluted emulsion. The measurement temperature is 25°C. Each sample is repeated 3 times.

[0042] The results are as follows Figure 4-5 As shown, the average droplet size of Pickering emulsion 1 is 273.6±15.4 nm, and the PDI is 0.21±0.03, which is consistent with the size range of nanoemulsions and has a narrow distribution; the Zeta potential is -35.6±2.1 mV, indicating that the emulsion has good electrostatic stability.

[0043] (2) Observation of the microstructure of the emulsion The microstructure and structure of Pickering emulsion 1 prepared in Example 2 were observed using a laser confocal microscope. Nile red (an oil-soluble fluorescent dye) and cationic dye Nile blue were used to stain the emulsion sample, respectively. The stained emulsion was dropped onto a glass slide, covered with a coverslip, and observed under a laser confocal microscope. By superimposing fluorescence signals from different channels, it was observed whether the oil droplets were completely wrapped by the interface layer, thus visually verifying the formation of the "cross-linked interface layer".

[0044] The results are as follows Figure 6 As shown, the circular area formed by green fluorescence (oil phase labeled with Nile Red) is tightly and completely surrounded by the bright ring formed by red fluorescence (interface layer labeled with Nile Blue). The two have a high degree of overlap, which intuitively and powerfully proves that a dense "cross-linked interface layer" has been successfully constructed on the surface of each oil droplet.

[0045] (3) Chemical structure analysis of the interface layer Accurately weigh 0.5 g of grape seed extract and zinc gluconate, dissolve them separately in 10 mL of deionized water, mix them, and stir thoroughly to ensure complete reaction. Centrifuge the reacted solution, collect the precipitate, and wash it three times with deionized water to remove unreacted substances. Then freeze-dry to obtain the zinc ion-grape seed extract coordination product. Simultaneously, freeze-dry the grape seed extract as a control group. Take 1 mg of dried Pickering emulsion 1 sample prepared in Example 2 and mix it with dried potassium bromide powder at a mass ratio of 1:100. Grind evenly and press into transparent sheets; use a Fourier transform infrared spectrometer at 4000-500 cm⁻¹. -1 Scan within the range, with a resolution of 4 cm. -1 By comparing and analyzing the shifts and changes in characteristic absorption peaks in the spectra of zinc ion-grape seed extract coordination products and grape seed extract, the formation of coordination bonds was confirmed.

[0046] The results are as follows Figure 7 As shown, compared with the zinc ion-crosslinked grape seed extract, the spectrum of the zinc ion-grape seed extract coordination model product exhibited a characteristic change: located at 1270 cm⁻¹ -1 The peak of CO stretching vibration at that location shifted to 1290 cm. -1 and at 1400 cm -1 A new absorption peak belonging to the Zn-O coordination bond appeared; this provides key chemical evidence for the construction of the "interfacial metal-phenol coordination network", while grape seed extract that is not cross-linked with zinc ions does not have this characteristic peak.

[0047] 2. Stability Evaluation (1) Storage stability study Pickering emulsion 1 prepared in Example 2 and comparative emulsions 1-3 prepared in Comparative Examples 1-3 were dispensed into transparent glass bottles and placed in a 4°C refrigerator, a 25°C constant temperature incubator, and a 55°C oven for accelerated stability testing. Samples were taken on days 0, 1, 3, 7, 14, and 30 to observe whether there was any layering, precipitation, oil separation, or discoloration in the appearance of the samples, and the particle size and PDI changes were measured using a laser particle size analyzer.

[0048] The results are as follows Figure 8 As shown, in the accelerated test at 55°C, the emulsion sample remained uniformly milky white for 60 days, without stratification or oil ring precipitation; the average particle size slowly increased from the initial 273.6 nm to 322.4 nm on day 60; after 60 days of storage at room temperature (25°C), its average particle size increased to 295.8 nm; under refrigerated conditions at 4°C, the particle size only slightly increased to 282.9 nm. It is evident that the Pickering emulsion 1 provided by this invention maintains excellent physical stability even under long-term storage.

[0049] The results are as follows Figure 9 As shown, the stability of contrast emulsions 1-2 in the accelerated test at 55℃ was significantly worse than that of Pickering emulsion 1 provided by this invention. The particle size growth rate of the zinc ion-free crosslinked emulsion (contrast emulsion 1) was significantly accelerated, with an increase of 62% within 30 days, indicating that the lack of crosslinking network resulted in insufficient mechanical strength of the interfacial layer, which could not effectively inhibit Ostwald ripening and aggregation of droplets. The initial particle size of the grape seed extract-free emulsion (contrast emulsion 2) was relatively large and continued to grow rapidly over time, proving that grape seed extract, as a key ligand, is crucial for constructing a dense and stable initial interfacial structure. The disordered physical mixing emulsion (contrast emulsion 3) experienced demulsification on the first day of storage (unable to be recorded), indicating that the "ginsenoside-grape seed extract binary self-assembled body" formed through specific steps is an important physical basis for constructing an effective particle-stable interfacial layer, and simple physical mixing cannot achieve basic stability of the emulsion.

[0050] (2) Centrifugation and environmental stability investigation Take 5 mL of each emulsion sample (Pickerling emulsion 1 prepared in Example 2 and control emulsions 1-3 prepared in Comparative Examples 1-3) and place them in a 10 mL centrifuge tube. Centrifuge at 8000 r / min for 15 min. After centrifugation, immediately observe whether the sample shows stratification, precipitation or floating oil layer, and determine whether there is precipitation at the bottom of the centrifuge tube. At the same time, take the upper layer of emulsion after centrifugation, re-measure its particle size, and calculate the particle size change rate.

[0051] Thermal stability test: 5 mL of emulsion (Pickerling emulsion 1 prepared in Example 2 and control emulsions 1-3 prepared in Comparative Examples 1-3) were placed in 10 mL sample bottles and heated in a 90°C water bath for 30 min, and then rapidly cooled to 25°C in an ice bath; the appearance and color changes of the emulsion before and after heating were observed, and its particle size was measured.

[0052] Freeze-thaw stability test: 5 mL of emulsion was frozen at -20°C for 24 hours and then thawed naturally at 25°C. This is one freeze-thaw cycle. This process was repeated 3 times. The state of the emulsion was observed after each freeze-thaw cycle, and its particle size was measured after the third cycle.

[0053] The results are as follows Figure 10 As shown, after centrifugation at 8000 r / min for 15 min, the Pickering emulsion 1 sample prepared in Example 2 did not show oil-water separation or bottom sedimentation. After three freeze-thaw cycles or heat treatment at 90℃, the Pickering emulsion 1 still maintained a basically uniform state, with only a slight increase in particle size (295 nm), but no demulsification phenomenon, indicating that its interface layer can effectively resist the impact of mechanical stress and temperature changes.

[0054] In contrast, the stability of the control emulsions 1-3 prepared in Comparative Examples 1-3 was significantly reduced. The zinc-free crosslinking group (control emulsion 1) showed oil-water separation after centrifugation, indicating insufficient mechanical strength of its interfacial layer; the grape seed extract-free group (control emulsion 2) showed a significant increase in particle size and precipitation after freeze-thaw cycles, reflecting poor interfacial integrity; while the physically mixed group (control emulsion 3) rapidly demulsified in all tests, indicating its inability to form an effective stable interface.

[0055] (4) Study on the chemical stability of active ingredients To systematically evaluate the long-term chemical protection capability of Pickering emulsion for active ingredients and to explore its differences from traditional emulsion systems in terms of active ingredient stability, a comparative study was conducted on the retention rate of total ginsenosides. Three groups of samples were set up: Pickering emulsion 1 prepared in Example 2, traditional Pickering emulsion prepared in Comparative Example 4, and a total ginsenoside solution (initial total ginsenoside concentration was the same as in Example 2). All samples were aliquoted and placed in a 40°C constant temperature incubator for accelerated stability testing. Samples were taken on days 0, 7, 14, 30, and 60. High-performance liquid chromatography (HPLC) combined with an evaporative light scattering detector or an ultraviolet detector was used to determine the total content of total ginsenosides in each group of samples. The total ginsenoside content on day 0 was taken as 100%, and the content retention rate at each time point was calculated to comprehensively evaluate the impact of different dosage forms on the overall chemical stability of the active ingredients.

[0056] The results are as follows Figure 11As shown, under accelerated storage conditions at 40°C, the retention rate of total ginsenosides in Pickering emulsion 1 prepared in Example 2 was consistently significantly higher than that of traditional Pickering emulsion and ginsenoside solution. Even after 60 days of storage, the retention rate of total saponins in Pickering emulsion 1 remained as high as 88.2% ± 1.6%; in contrast, the retention rate of traditional Pickering emulsion prepared in Comparative Example 4 decreased to 66.7% ± 2.5%, and the ginsenoside solution group only reached 44.3% ± 3.0%. This demonstrates that the "metal-phenol network" interface layer constructed based on the coordination crosslinking of zinc ions and grape seed extract effectively blocks environmental factors such as water and oxygen, providing excellent long-term chemical stability protection for total ginsenosides. This confirms the "protective shield" effect of the Pickering emulsion system from the perspective of active ingredient retention.

[0057] 3. pH responsiveness evaluation (1) Study on in vitro release behavior Using the dialysis bag diffusion method, an emulsion equivalent to 10 mg of ginsenosides (Pickerling emulsion 1 prepared in Example 2 and control emulsions 1-3 prepared in Comparative Examples 1-3) was accurately measured and placed into a dialysis bag with a molecular weight cutoff of 3500 Da. The ends were tied tightly, and the bag was placed in an Erlenmeyer flask containing 50 mL of release medium. The medium was phosphate buffer solution at pH 7.4 (simulating storage conditions) and acetate-sodium acetate buffer solution at pH 5.5 (simulating the weakly acidic conditions of the skin surface). The Erlenmeyer flask was placed in a constant temperature shaker at 37°C and shaken at a rate of 100 r / min. At predetermined time points (e.g., 5, 15, 30, 60, 120, 240, 480, 720 min), 1 mL of release medium was taken out of the Erlenmeyer flask (and 1 mL of fresh medium at the same temperature was added at the same time). The concentration of total ginsenosides in the medium was determined by high performance liquid chromatography, the cumulative release rate was calculated, and the release curve was plotted.

[0058] The results are as follows Figure 12 As shown, in a medium of pH 7.4, the cumulative release rate of ginsenosides in Pickering emulsion 1 prepared in Example 2 was less than 25% within 24 hours, indicating that the interfacial layer structure remained intact under storage conditions, effectively locking in the active ingredients. In a medium of pH 5.5, the release accelerated significantly, exceeding 35% within 30 minutes and reaching over 70% within 2 hours, demonstrating rapid and thorough pH-responsive release characteristics. In contrast, the control emulsions 1-3 prepared in Comparative Examples 1-3 showed no pH responsiveness, with release curves highly overlapping in both pH media. Specifically, the zinc ion-free crosslinking group (control emulsion 1) exhibited passive, medium-speed diffusion; the grape seed extract-free group (control emulsion 2) released faster due to poor interfacial stability; and the physically mixed group (control emulsion 3) experienced rapid burst release due to encapsulation failure.

[0059] (2) Response mechanism verification Atomic absorption spectroscopy analysis: Pickering emulsion 1 prepared in Example 2 was mixed with equal volumes of buffer solutions at pH 7.4 and pH 5.5, respectively. After incubation for 2 hours, the supernatant was collected by high-speed centrifugation, and the free Zn²⁺ in the supernatant was determined using an atomic absorption spectrometer. + The concentrations were compared to assess the stability of coordination bonds under different pH conditions.

[0060] AAS measurement results are as follows Figure 13 As shown, after incubation at pH 5.5, the free Zn²⁺ in the supernatant of Pickering emulsion 1 prepared in Example 2 was [data missing]. + The concentration was significantly higher than that under pH 7.4 conditions, confirming that the acidic environment promoted the breaking of coordination bonds and the dissociation of zinc ions.

[0061] 4. Functional evaluation (1) Determination of active ingredient encapsulation rate Accurately weigh 2 g of emulsion (Pickerling emulsion 1 prepared in Example 2, and comparative emulsions 1-3 prepared in Comparative Examples 1-3), using vitamin C solution as a positive control, place in a centrifuge tube, and centrifuge at 4°C and 10000 r / min for 30 min; carefully aspirate the lower aqueous phase, filter through a 0.22 μm microporous membrane, and determine the concentration of free ginsenosides in the aqueous phase using high performance liquid chromatography; the encapsulation rate is calculated according to the following formula: encapsulation rate (%) = (total amount of ginsenosides fed - amount of free ginsenosides) / total amount of ginsenosides fed × 100%.

[0062] The results are shown in Table 1. The encapsulation rate of ginsenosides in Pickering emulsion 1 reached 82.4% ± 3.1%, indicating that the system has efficient drug loading capacity and stable encapsulation performance, which is beneficial to the preservation and delivery of active ingredients.

[0063] Table 1

[0064] (2) Evaluation of in vitro antioxidant performance The test emulsions (Pickerling emulsion 1 prepared in Example 2, and control emulsions 1-3 prepared in Comparative Examples 1-3, with vitamin C solution as the positive control) were appropriately diluted with anhydrous ethanol and mixed with an equal volume of 0.1 mM DPPH ethanol solution. The mixture was reacted in the dark for 30 min, and the absorbance of the mixture was measured at a wavelength of 517 nm. Anhydrous ethanol was used as a blank, and vitamin C was used as a positive control. The clearance rate was calculated using the formula: Clearance rate (%) = [1 - (Asample - Abackground) / Acontrol] × 100%.

[0065] The results are as follows Figure 14-15 As shown, the free radical scavenging rate of Pickering emulsion 1 was significantly higher than that of control emulsions 1-3; the IC50 of Pickering emulsion 1 was...50 The value was significantly lower than that of the control group without cross-linking (comparison emulsion 1) and without grape seed extract (comparison emulsion 2), confirming that ginsenosides and grape seed extract produced a significant synergistic antioxidant enhancement effect in the presence of a metal-phenol network, thereby improving the overall antioxidant activity of the system.

[0066] (3) Preliminary investigation of skin application performance Moisturizing performance test: Healthy volunteers were recruited, and test areas were marked on the inner forearm. Pickering lotion 1 prepared in Example 2 was applied to one side, and commercially available moisturizing products 1-2 (a blank control without active ingredients, purchased from the Tmall official flagship store) were applied to the other side. The moisture content of the stratum corneum of the skin in each area was measured using a skin moisture meter before application and at 1 h, 2 h, 4 h, and 8 h after application, and the moisturizing rate was calculated.

[0067] Skin irritation test: Acute skin irritation test was conducted on rabbits in accordance with the "Cosmetic Safety Technical Specifications". The hair on both sides of the spine of the rabbits (purchased from Changchun Yisi Experimental Animal Technology Co., Ltd.) was removed. Pickering emulsion 1 prepared in Example 2 was applied to the left side, and commercially available moisturizing products 1-2 were applied to the right side as a control. After 4 hours of occlusion, the test products were removed, and the skin reactions such as erythema and edema after 1 hour, 24 hours, 48 ​​hours and 72 hours were observed and recorded. The Draize score was calculated.

[0068] Cytotoxicity assay: Human immortalized epidermal keratinocytes (HaCaT) were used to evaluate cytotoxicity, aiming to systematically assess the biosafety of Pickering emulsion. Pickering emulsion 1 prepared in Example 2, conventional Pickering emulsion prepared in Comparative Example 4, and blank Pickering emulsion prepared in Comparative Example 5 were diluted with cell culture medium to different concentrations (0.1, 0.25, 0.5, 1.0 mg / mL) to prepare sample extracts. HaCaT cells were seeded in 96-well plates and cultured for 24 h. The culture medium was then replaced with sample extracts of Pickering emulsion at different concentrations (0.1, 0.25, 0.5, 1.0 mg / mL), and cultured for another 24 h. CCK-8 solution was added to each well, and after incubation for a certain period, the absorbance at 450 nm was measured using a microplate reader, and the relative cell viability was calculated.

[0069] The results are as follows Figure 16-18As shown, skin tests on volunteers revealed that after applying the Pickering emulsion prepared in Example 2 for 18 hours, the moisture content of the stratum corneum remained significantly increased, rising by 13.7% compared to before application, demonstrating excellent sustained moisturizing performance. The acute skin irritation test on rabbits showed a Draize score of 0, with no adverse reactions such as erythema or edema observed, indicating that the emulsion is non-irritating to the skin. Cytotoxicity tests showed that within the concentration range of 0.1-1.0 mg / mL, the HaCaT cell survival rate of the Pickering emulsion prepared in Example 2 was consistently higher than 87.6%, exhibiting a significant advantage compared to traditional surfactant (Tween 80) emulsions, where the cell survival rate dropped to 58.9% at 1.0 mg / mL, demonstrating significant concentration-dependent toxicity. Therefore, the Pickering emulsion provided by this invention not only possesses outstanding moisturizing efficacy but also exhibits significant safety advantages compared to traditional surfactant systems, providing experimental evidence for its use as a gentler and safer transdermal delivery system.

[0070] 5. Rheological property characterization (1) Steady-state shear test (flow behavior) The Pickering emulsion sample 1 prepared in Example 2 was uniformly loaded onto the parallel plate fixture (plate diameter 40 mm, gap 1.0 mm) of the rheometer. The test was conducted at a constant temperature of 25°C. First, strain scanning was performed to determine the linear viscoelastic region of the sample; then, within the determined linear region, steady-state flow scanning was performed, with shear rates starting from 0.1 s⁻¹. -1 Linearly increase to 100 s -1 Record the curve of apparent viscosity as a function of shear rate.

[0071] The results are as follows Figure 19 As shown, Pickering emulsion 1 exhibits typical shear-thinning behavior, meaning its apparent viscosity decreases significantly with increasing shear rate; at low shear rates (0.1 s⁻¹), the viscosity remains relatively stable. -1 At this temperature, the emulsion exhibits a high viscosity, which is beneficial for its stability during static storage and prevents rapid sedimentation or aggregation of droplets. As the shear rate increases to the range of the simulated application process (10-100 s⁻¹), the emulsion becomes more stable. -1 The viscosity drops rapidly, indicating that the Pickering emulsion 1 provided by the present invention becomes easy to flow when subjected to external forces (such as application or pumping), and has good application feel and processing adaptability.

[0072] (2) Dynamic oscillation test (viscoelastic properties) Under the same fixture and temperature conditions, dynamic frequency scanning was performed on Pickering emulsion 1 prepared in Example 2 and comparative emulsions 1-3 prepared in Comparative Examples 1-3, respectively. A fixed oscillatory strain (typically 1%) was applied within the defined linear viscoelastic region, and the angular frequency was scanned from 0.1 rad / s to 100 rad / s. The instrument automatically recorded and output the curves of storage modulus (G′, representing the elastic or solid behavior of the sample) and loss modulus (G″, representing the viscous or liquid behavior of the sample) as a function of angular frequency.

[0073] The results are as follows Figure 20 As shown, throughout the entire frequency range of the test, the storage modulus (G′) of the emulsion was consistently significantly higher than the loss modulus (G″), and the two modulus curves were nearly parallel, exhibiting weak frequency dependence. This characteristic is a clear hallmark of solid-like gel networks; it directly demonstrates that the "metal-phenol coordination network" constructed at the interface by zinc ions and grape seed extract not only forms a barrier on the surface of individual droplets but also forms a continuous and stable three-dimensional elastic network structure throughout the entire emulsion system. This effectively locks oil droplets in their respective positions, resisting deformation and aggregation, and mechanically explains the excellent physical stability exhibited by the emulsion (such as resistance to centrifugation, freeze-thaw resistance, and long-term storage stability). The rheological behavior of the comparative emulsions 1-3 prepared in Comparative Examples 1-3 differs from this. The G′ value of the emulsion without zinc ion crosslinking is significantly reduced, and the network strength is insufficient; the emulsion without grape seed extract exhibits a viscous liquid (G″>G′); while the disordered physical mixture emulsion shows almost no elastic response. The results demonstrate that the synergistic effect of zinc ion crosslinking, grape seed extract, and the ordered self-assembly process is a prerequisite for network construction.

[0074] 6. In vitro percutaneous permeation test The Franz diffusion cell method was used. Freshly separated pigskin (after dehairing and cleaning, with the stratum corneum facing upwards) was fixed between the supply and receiving cells. A pH 7.4 phosphate buffer solution containing 1% Tween 80 was added to the receiving cell as the receiving medium to remove air bubbles under the skin. 200 mg of Pickering emulsion 1 prepared in Example 2 was added to the supply cell, with ginsenoside solution as the control. The entire system was maintained in a constant temperature water bath at 37°C. The receiving cell was stirred at a uniform speed of 500 r / min using a magnetic stirrer. At predetermined time points (2, 4, 8, 12, 24, 36, and 48 h), 1 mL of sample was taken from the receiving cell, and an equal volume of fresh receiving solution (pH 7.4 phosphate buffer solution containing 1% Tween 80) was added simultaneously. The concentration of ginsenosides was determined using HPLC, and the cumulative permeation per unit area was calculated (as shown in Table 2). After the experiment, the skin was recovered, the surface was cleaned, and the skin was cut into small pieces and homogenized. The amount of active ingredients retained in the skin was extracted and measured, and the skin retention rate was calculated.

[0075] The results are as follows Figure 21-22As shown in the Franz diffusion cell experiment, the cumulative permeation per unit area of ​​Pickering emulsion 1 prepared in Example 2 was significantly higher than that of ginsenoside solution within 48 h. Simultaneously, the amount of drug retained in the skin was also higher, indicating that this system not only promotes transdermal absorption of active ingredients but also forms a certain reservoir effect in the local skin, which is beneficial for the long-term effect.

[0076] Table 2

[0077] Note: Table 2 shows the cumulative permeability of ginsenosides per unit area at different time points, in μg / cm². 2 Mean ± SD, n=3.

[0078] The specific embodiments of the present invention disclosed above are only for illustrating the present invention. These specific embodiments do not describe all details exhaustively, nor do they limit the invention to only the described embodiments. Many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the present invention, thereby enabling those skilled in the art to better understand and utilize the present invention.

Claims

1. A method for preparing a triple-assembled Pickering emulsion loaded with ginsenosides, characterized in that, The preparation method includes the following steps: S1: Weigh out ginsenosides and dissolve them in anhydrous ethanol, then heat to aid dissolution, to obtain solution A; weigh out grape seed extract and dissolve it in deionized water preheated to 45±2℃, to obtain solution B; under magnetic stirring at 400 rpm, add solution A dropwise to solution B, then place the beaker in a 45℃ constant temperature water bath and continue stirring for 30-40 min, then cool to room temperature to obtain a binary self-assembled suspension; S2: Weigh squalane and tocopherol into a beaker, place the beaker in a 40°C water bath, and stir until the mixture is completely homogeneous and transparent to obtain the oil phase; S3: The binary self-assembled suspension prepared in S1 was used as the aqueous phase and transferred to the processing cup of a high-speed shear emulsifier for emulsification at 12,000 rpm. Under high-speed shear conditions, the preheated oil phase prepared in S2 was added dropwise to the aqueous phase at a uniform rate for 5 min. After the addition was completed, high-speed shear emulsification was continued at 12,000 rpm for 15 min to obtain the primary pre-emulsion. S4: Weigh zinc gluconate and dissolve it in deionized water to obtain a zinc ion solution; under continuous stirring at 500 rpm, add the zinc ion solution dropwise to the primary pre-emulsion obtained in S3 for 5 min; after the addition is complete, continue stirring for 30 min to obtain the intermediate pre-emulsion. S5: The intermediate pre-emulsion prepared in S4 was transferred to a high-pressure homogenizer and homogenized for 3 cycles at a pressure of 60 MPa. After homogenization, the pH was adjusted with 0.1 mol / L sodium hydroxide solution, and then the preservative ketone was added. Deionized water was added to obtain an emulsion mixture. The emulsion mixture was stirred at low speed at 200 rpm for 60 min at room temperature to obtain a triple-assembled Pickering emulsion.

2. The production method according to claim 1, characterized by, The mass-to-volume ratio of ginsenosides and anhydrous ethanol described in S1 is 100 mg: 2 mL.

3. The production method according to claim 1, characterized by, The mass-to-volume ratio of grape seed extract and deionized water described in S1 is 100 mg: 68 mL.

4. The method of claim 1, wherein, The mass ratio of squalane and tocopherol in S2 is 8:0.

5.

5. The preparation method according to claim 1, characterized in that, The mass ratio of zinc gluconate and deionized water in S4 is 0.5:

10.

6. The method of claim 1, wherein, The pH value described in S5 is adjusted to 7.0 ± 0.

1.

7. The preparation method according to claim 1, characterized in that, The amount of the preservative ketone mentioned in S5 is 0.1% of the total mass of the Pickering emulsion.

8. A triple-assembled Pickering emulsion loaded with ginsenosides, characterized in that, The triple-assembled Pickering emulsion is obtained by the preparation method described in any one of claims 1 to 7.

9. The use of the triple-assembled Pickering emulsion according to claim 8 in the preparation of anti-aging cosmetics.

10. The use of the triple-assembled Pickering emulsion according to claim 8 in the preparation of transdermal drug delivery formulations.

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