A core-shell nanoparticle of polygalactoside-aescin sodium, its preparation method and application

Abstract

This invention relates to novel materials and their applications in cosmetics, specifically disclosing a polygalactosin-sodium aescinate core-shell nanoparticle, its preparation method, and its applications. The nanoparticles use polygalactosin as the hydrophobic core component and sodium aescinate as the hydrophilic shell component. They are prepared via an antisolvent precipitation-high shear dispersion coupling process, allowing polygalactosin to rapidly nucleate in an aqueous phase and be coated with sodium aescinate to form core-shell structured nanoparticles. Subsequently, the organic solvent is removed by vacuum rotary evaporation, followed by washing and freeze-drying to obtain the nanoparticles. The nanoparticles obtained by this invention exhibit controllable particle size distribution and good dispersibility, improving the dispersion stability of polygalactosin in aqueous systems. They also demonstrate inhibitory activity against tyrosinase in tyrosinase inhibition experiments and free radical scavenging ability in DPPH and ABTS free radical scavenging experiments, making them suitable for pharmaceutical, cosmetic formulation, and cosmetic applications related to skin whitening and anti-oxidation.

Description

Technical Field

[0001] This invention relates to new materials and their application in cosmetics, specifically to a core-shell nanoparticle of polygalactoside-aescin sodium, its preparation method, and its application. Background Technology

[0002] In the field of topical skincare, the stability and antioxidant capacity of active ingredients are crucial for addressing skin problems caused by external environmental factors. With accelerated industrialization and the impact of environmental changes, ozone layer depletion has led to increased ultraviolet (UV) radiation, making UV exposure one of the primary external factors causing photoaging. UV radiation can induce the production of large amounts of reactive oxygen species (ROS) in the skin, accelerating melanin synthesis and damaging skin structure, resulting in dull skin tone, age spots, and a decline in skin barrier function, making facial photoaging more prevalent and severe. Simultaneously, with the gradual improvement of socioeconomic levels and living standards, people are paying increasing attention to skin health and appearance, demanding safe, gentle topical skincare products with whitening and antioxidant functions. However, existing whitening and antioxidant products generally suffer from insufficient stability of active ingredients, limited efficacy, and poor transdermal penetration, making it difficult to simultaneously ensure safety, stability, and long-term effectiveness.

[0003] Polydatin (PD) is a natural active ingredient derived from plants such as Polygonum cuspidatum. It has significant whitening and antioxidant effects, but its solubility and dispersion stability in water are limited, and it is very easy to precipitate or lose its activity, which limits its application and efficacy in topical skin preparations (such as serums, lotions, gels, masks, etc.).

[0004] Sodium aescinate (SA) is a natural triterpenoid saponin extracted from plants of the Aesculus family. It possesses pharmacological activities such as anti-inflammatory, anti-swelling, microcirculation promotion, and skin barrier repair, making it particularly suitable for topical applications to soothe sensitive skin, improve redness, and reduce swelling. It exhibits certain surface activity, film-forming properties, and stabilizing effects, and can function as a structural stabilizer and interface regulator in aqueous formulations. However, its large molecular weight, weak lipid solubility, and poor permeability to the stratum corneum, coupled with its tendency to aggregate in aqueous formulations leading to low bioavailability and fluctuating efficacy, also limit its application in formulations.

[0005] In existing technologies, to improve the availability of hydrophobic or poorly soluble active ingredients in aqueous systems, methods such as solubilizers, emulsifiers, or nano-sizing are commonly used. However, these methods often suffer from problems such as narrow process windows, difficulty in controlling particle size stably, high dependence on skin-irritating excipients, or insufficient consistency in large-scale production.

[0006] Therefore, there is an urgent need to develop a new type of nanoparticle product that is highly efficient, stable, and combines whitening and anti-oxidation properties, while also being simple and scalable to improve the stable dispersion of polysaccharide in aqueous systems and overcome the shortcomings of sodium aescinate in topical application, such as poor permeability and unstable efficacy. This product can then be applied to the skincare product manufacturing field to meet the application needs of skin whitening and anti-oxidation. Summary of the Invention

[0007] The primary objective of this invention is to prepare core-shell nanoparticles using polygalactosidase as the hydrophobic core component and sodium aescinate as the hydrophilic shell component via an antisolvent precipitation-high shear dispersion coupling process. This process allows polygalactosidase to rapidly nucleate in an aqueous phase and be coated with sodium aescinate to form core-shell nanoparticles. Subsequently, the organic solvent is removed by vacuum rotary evaporation, followed by washing and lyophilization to obtain the nanoparticles. The nanoparticles obtained by this invention exhibit controllable particle size distribution and good dispersibility, improving the dispersion stability of polygalactosidase in aqueous systems. Furthermore, they demonstrate tyrosinase inhibition in tyrosinase experiments and free radical scavenging ability in DPPH and ABTS free radical scavenging experiments, making them suitable for pharmaceutical, cosmetic formulations, and cosmetic applications related to skin whitening and anti-oxidation.

[0008] The second objective of this invention is to provide a method for preparing the above-mentioned polygalactoside-aescin sodium core-shell nanoparticles PD-SA NPs.

[0009] A third objective of this invention is to provide the application of the above-mentioned polygalactoside-aescin sodium core-shell nanoparticles PD-SA NPs in the preparation of skin whitening and antioxidant products, including but not limited to topical skin preparations such as serums, lotions, gels, and masks.

[0010] To achieve the above objectives, the present invention specifically adopts the following technical solution: The product provided by this invention, polydipsia-aescin sodium core-shell nanoparticles, has polydipsia as the core and aescin sodium as the shell material coating the outside of polydipsia, forming a core-shell nanostructure with a particle size range of 50-200 nm. The mass ratio of polydipsia to aescin sodium is 1:1.2-2.8, more preferably 1:1.6-2.0, and most preferably 1:1.6-1.8. Within the above mass range, the final nanoparticle solution has a more uniform particle size and a more complete core-shell spherical structure with a deeper inner core and a shallower outer core, resulting in better long-term stability.

[0011] The inventors further provided a method for preparing the above-mentioned polygalactoside-aescin sodium core-shell nanoparticles PD-SA NPs, the steps of which are as follows: (1) Dissolve polydipsia glycoside in a mixed solvent of acetone and ethanol to obtain a polydipsia glycoside organic phase solution; (2) Dissolve sodium aescinate in deionized water to obtain an aqueous solution of sodium aescinate; (3) Under high shear stirring conditions, the organic phase solution of Polygonum cuspidatum was added dropwise to the aqueous phase solution of sodium aescinate to form a core-shell nanoparticle dispersion system; (4) The organic solvent was removed by a rotary evaporator under reduced pressure to obtain an aqueous dispersion of polysaccharide-aescin sodium; (5) The above polydipsia-aescin sodium aqueous dispersion was freeze-dried to obtain polydipsia-aescin sodium core-shell nanoparticles.

[0012] The more specific steps are as follows: S1: Preparation of the organic phase of polygalactoside: Weigh polygalactoside and dissolve it in an organic solvent to obtain a polygalactoside organic phase solution; preferably, the mass concentration of the above solution is 1-8 mg / mL, and in specific applications, 1 mg / mL, 4 mg / mL or 8 mg / mL is preferred; S2: Preparation of sodium aescinate aqueous phase: Weigh sodium aescinate, dissolve sodium aescinate in deionized water, and magnetically stir at 20-35℃ and 800-1000rpm for 30-40min, followed by ultrasonic treatment for 10-20min to obtain sodium aescinate aqueous phase solution; preferably, the mass concentration of the above solution is 1-8mg / mL, and in specific applications, 1mg / mL, 4mg / mL, or 8mg / mL are preferred; S3: Dropping and High-Shear Dispersion: Place the sodium aescinate aqueous solution in a high-shear mixer container, and set the high-shear mixer speed to 10000-13000 rpm; add the polygalactosyl glycoside organic solution to the above sodium aescinate aqueous solution at a dropping rate of 1-3 mL / min, and maintain high-shear stirring during the dropping process; The amount of polygalactoside organic phase solution added is determined based on the mass ratio of polygalactoside to sodium aescinate of 1:1.2-2.8. It is only necessary to ensure that the mass ratio of the two in the final product is within the above range. After the addition is completed, continue high-shear stirring for 5-15 minutes to obtain a homogeneous emulsion-dispersion system. S4: Removal of organic solvents: The obtained dispersion system was transferred to a rotary evaporator under reduced pressure and evaporated under reduced pressure in a water bath at 25-45℃ to remove acetone and ethanol, and PD-SA NPs aqueous dispersion was obtained. S5: Purification: The PD-SA NPs aqueous dispersion was separated by centrifugation or ultrafiltration, resuspended in deionized water and washed 2-5 times; finally, the sample was freeze-dried to obtain polydipsia glycoside-aescin sodium core-shell nanoparticles, which were in the form of off-white powder solid.

[0013] In step S1, the polygalactosodium glutamate used was purchased from Shanghai Ron, with the catalog number R018556; the organic solvent used in this step is a mixed solvent of acetone and ethanol in a volume ratio of 1:1-3.

[0014] In S2, sodium aescinate was purchased from Shanghai Ron, with the product number R023552; the ultrasonic treatment was performed at a frequency of 40kHz and a power of 30%-80%.

[0015] The stirring conditions of the dropwise addition process described in S3 can be adjusted as needed to obtain core-shell nanoparticles with stable particle size and good dispersibility.

[0016] The prepared nanoparticles were resuspended in phosphate-buffered saline (PBS, pH 7.4, 0.01 M) to form a dispersion with a final concentration of PD-SA NPs of 10 mg / mL. The dispersion was stored at room temperature (25℃) in the dark for 21 days before observation. The results showed that the particle size of all samples with a mass ratio of polygalactoside to sodium aescinate of 1:1.2–2.8 remained around 50–200 nm. However, during this period, some nanoparticle solutions with mass ratios of polygalactoside to sodium aescinate of 1:1.2–1:1.4 and 1:2.2–1:2.8 showed drug precipitation and sedimentation, while the nanoparticle solutions with a mass ratio of polygalactoside to sodium aescinate of 1:1.6–2.0 remained clear without turbidity or precipitation. Transmission electron microscopy revealed that when the mass ratio of polygalactoside to sodium aescinate was 1:1.6-1.8, the nanoparticles in the solution exhibited more uniform particle size and a more complete core-shell spherical structure with a deeper inner core and a shallower outer shell. This indicates that nanoparticle solutions within this ratio range possess better long-term stability.

[0017] The obtained core-shell nanoparticles with a mass ratio of 1:1.8 of polygalactoside and sodium aescinate were identified. Transmission electron microscopy (TEM) revealed core-shell characteristics in the morphology of the PD-SA NPs. Malvern particle size / potential analyzer was used to determine the particle size distribution at the zeta potential. Fourier transform infrared spectroscopy was used to detect the covalent bond peak changes of the PD-SA NPs. Near-spherical nanoparticles were observed under TEM, with some particles exhibiting a contrasting "dark core-light shell" characteristic, suggesting the formation of a core-shell structure.

[0018] In addition, the present invention further claims the application of the above-mentioned polygalactoside-aescin sodium core-shell nanoparticles in cosmetics, specifically for the preparation of skin whitening and antioxidant products, including but not limited to serums, lotions, gels, masks and other topical skin preparations.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: The core-shell nanoparticle structure is constructed using natural active ingredients, which improves the dispersion stability of polygalactoside while reducing the dependence on irritating excipients. The prepared polygalactoside-aescin sodium core-shell nanoparticles PD-SA NPs have the advantages of structural stability, relatively simple preparation process and scalability, which can meet the application needs of skin whitening and anti-oxidation, and have good application prospects. Attached Figure Description

[0020] Figure 1 A schematic diagram of the preparation route for PD-SA NPs; Figure 2 The results of the lyophilized powder morphology of PD-SA NPs (left figure) and water solubility test (right figure); Figure 3 These are the transmission electron microscopy results of PD-SA NPs. The left image shows the results of low-magnification transmission electron microscopy, and the right image shows the results of high-magnification transmission electron microscopy. Figure 4 The nanoparticle size distribution of PD-SA NPs is shown. Figure 5 A zeta potential analysis plot of PD-SA NPs; Figure 6 Fourier transform infrared spectra of sodium aescinate, polygalactoside, and PD-SA NPs; Figure 7 The in vitro release rate of PD-SA NPs (%); Figure 8 The inhibition rate of PD-SA NPs against tyrosinase (%). Figure 9 The time curves showing the inhibition of tyrosinase activity by different concentrations of PD-SA NPs; Figure 10 IC50 of PD-SA NPs against tyrosinase inhibitory activity 50 value; Figure 11 Time curves showing the inhibition of tyrosinase activity by different concentrations of kojic acid; Figure 12 IC50 of kojic acid's inhibitory activity against tyrosinase 50 value; Figure 13 DPPH radical scavenging rate of PD-SA NPs (%) Figure 14 ABTS radical scavenging rate (%) of PD-SA NPs. Detailed Implementation

[0021] To illustrate the technical content, objectives, and effects of this invention in detail, the following specific embodiments further explain the invention. Based on the embodiments of this invention, any equivalent substitutions or modifications made by those skilled in the art without any creative effort should be included within the scope of protection of this invention.

[0022] Example 1: Preparation method, identification method and identification results of polygalactoside-aescin sodium core-shell nanoparticles (PD-SA NPs) 1.1 Preparation method (antisolvent precipitation method, such as...) Figure 1 (As shown) (1) Preparation of the organic phase of polydipsia: Polydipsia was weighed and dissolved in a mixed solvent of acetone and ethanol in a volume ratio of 1:3 to obtain a clear or homogeneous organic phase solution with concentrations of 1 mg / mL, 4 mg / mL and 8 mg / mL, respectively.

[0023] (2) Preparation of sodium aescinate aqueous phase: Weigh sodium aescinate and dissolve it in deionized water; stir magnetically at 800-1000 rpm for 30-40 min at 20-35℃ to promote dissolution; then perform ultrasonic treatment for 10-20 min to further homogenize (frequency 40kHz, power 30%-80%, if necessary, use an ice bath to control the temperature at 20-35℃).

[0024] More specifically in this embodiment, magnetic stirring at 1000 rpm for 40 min at room temperature was used to promote dissolution; then ultrasonic treatment was performed for 15 min to further homogenize (frequency 40 kHz, power 50%, while using an ice bath to control the temperature at 20 °C); thus obtaining aqueous solutions of sodium aescinate with concentrations of 1 mg / mL, 4 mg / mL, and 8 mg / mL.

[0025] (3) Dropping and high shear dispersion: Place the sodium aescinate aqueous phase in the container of the high shear mixer, turn on the high shear mixer and set the speed to 12000 rpm; drop the polygalactosodium organic phase into the aqueous phase at a rate of 1-3 mL / min, and keep the high shear stirring during the dropping process; The amount of polygalactoside organic phase solution added is determined based on the mass ratio of polygalactoside to sodium aescinate of 1:1.2-2.8. It is only necessary to ensure that the mass ratio of the two in the final product is within the above range. After the addition is completed, continue high-shear stirring for 15 minutes to obtain a homogeneous emulsion-dispersion system.

[0026] (4) Solvent evaporation: The obtained dispersion system was transferred to a rotary evaporator under reduced pressure and evaporated under reduced pressure in a water bath at 40°C to remove acetone and ethanol until no acetone and ethanol could be detected in the system, thus obtaining an aqueous dispersion of PD-SA NPs.

[0027] (5) Washing and freeze-drying: The PD-SA NPs aqueous dispersion was separated by centrifugation or ultrafiltration, resuspended in deionized water and washed repeatedly 2-5 times; freeze-drying (pre-freezing stage: -40℃, 4h; first drying: -70℃, 20Pa, 12h; second drying: 20℃, 10Pa, 6h) to obtain PD-SA NPs core-shell nanoparticles, which are in a powder-like solid state. By adjusting the amount of polygalactosin organic phase added and the concentrations of sodium aescinate aqueous phase and polygalactosin organic phase, the mass ratio of polygalactosin to sodium aescinate in the finally obtained PD-SA NPs core-shell nanoparticles was controlled to be 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1:2.0, 1:2.2, 1:2.4, 1:2.6, 1:2.8.

[0028] Screening and Optimization of Formulations: The nanoparticles obtained above at different mass ratios were resuspended in phosphate buffer (PBS buffer, pH=7.4, 0.01M) to form dispersions. The final concentration of PD-SA NPs in the dispersions was 10 mg / mL. The dispersions were stored at room temperature (25℃) in the dark for 21 days before observation. The results showed that the particle size of all samples remained around 50-200 nm. However, some nanoparticle solutions with a mass ratio of polygalactoside to sodium aescinate of 1:1.2-1:1.4 and 1:2.2-1:2.8 showed drug precipitation and sedimentation, while the nanoparticle solutions with a mass ratio of polygalactoside to sodium aescinate of 1:1.6-2.0 remained clear throughout, without turbidity or precipitation. Transmission electron microscopy comparison revealed that when the mass ratio of polygalactoside to sodium aescinate was 1:1.6-1.8, the particle size in the nanoparticle solution was relatively more uniform, and it also had a more complete core-shell spherical structure with a deeper inner core and a shallower outer core (e.g., ...). Figure 3 As shown, the mass ratio of polygalactoside to sodium aescinate is 1:1.8. This indicates that the nanoparticle solution within the above ratio range exhibits better long-term stability.

[0029] The PD-SA NPs core-shell nanoparticles used in the various identification and experimental examples below are all nanoparticles with a weight ratio of 1:1.8.

[0030] 1.2 Identification Methods The morphology and core-shell structure of PD-SA NPs were observed using transmission electron microscopy (TEM); the particle size distribution and zeta potential were determined using a Malvern particle size / potential analyzer; and the peak changes of covalent bonds in PD-SA NPs were detected using Fourier transform infrared spectroscopy.

[0031] 1.3 Identification Results The obtained PD-SA NPs aqueous dispersion was a colorless liquid, which turned into a white powder after lyophilization. The PD-SA NPs lyophilized powder dissolved in physiological saline resulted in a colorless and transparent solution. Figure 2Near-spherical nanoparticles were observed under TEM, exhibiting a contrasting "dark core-light shell" feature, suggesting the formation of a core-shell structure. Figure 3 Particle size analysis showed that the particle diameter was 50-200 nm, and the distribution was relatively concentrated. Figure 4 The zeta potential indicates that the particles are negatively charged, stabilizing between -15 and -20 mV. Figure 5 The characteristic absorption peaks of both polygalactosin and sodium aescinate can be observed simultaneously in the FTIR spectrum, and peak shifts or shape changes can be seen in the hydroxyl-carbonyl related region, suggesting that the two have hydrogen bond interactions and form a stable complex system. Figure 6 The above results collectively demonstrate that this embodiment successfully prepared polygalactoside-aescin sodium core-shell nanoparticles PD-SA NPs.

[0032] Experimental Example 1: In vitro release experiment of PD-SA NPs 1. Experimental Principle In vitro drug release assays are a classic method for evaluating the controlled-release properties of nanoparticles. By simulating physiological release conditions (0.01M PBS buffer), the cumulative release rate of the drug at different time points is measured to reflect the release kinetics of the formulation. This experiment used the dialysis bag method, placing PD-SANPs with a mass ratio of 1:1.8 of polygalactoside and sodium aescinate in the dialysis medium. The drug concentration in the dialysis fluid was measured at regular intervals, and the cumulative release rate was calculated to evaluate the sustained-release effect of nanoparticles on PD and SA and the differences in their release behavior.

[0033] 2. Experimental Preparation PBS buffer (pH=7.4, 0.01M, containing 3% ethanol by volume), dialysis bags, constant temperature water bath shaker, high performance liquid chromatograph, pipette, EP tubes, PD solution, SA solution, PD-SA NPs solution, etc.

[0034] 3. Experimental Grouping Set up a free PD group (PD solution), a free SA group (SA solution), a PD-SA NPs solution group (detecting PD release), and a PD-SA NPs solution group (detecting SA release).

[0035] 4. Experimental Procedure (1) Weigh a certain amount of PD, SA and PD-SA NPs powder, dissolve them in PBS buffer and prepare a 10 mg / mL solution; (2) Pretreatment of dialysis bags: Boil the dry dialysis bags in boiling water for 10 minutes, then rinse them with deionized water and set aside. (3) Sample preparation: Take equal amounts of PD solution, SA solution and PD-SA NPs solution and put them into the pretreated dialysis bag respectively, clamp both ends to ensure no leakage; (4) Release experiment: Place the dialysis bag into a centrifuge tube containing an appropriate amount of release medium and incubate it in a constant temperature water bath shaker at 37°C and 100 rpm. (5) Timed sampling: 1 mL of release medium was taken at set time points (0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 24, 48, 72, 96, 120, 144, 168 (h)) and 1 mL of fresh release medium was added at the same time to maintain a constant volume; (6) Content determination: After processing, the concentrations of PD and SA were determined by high performance liquid chromatography. (7) Calculate the cumulative release rate using the following formula. Where Cn is the drug concentration at the nth time point, V is the total volume of the release medium, Vi is the volume of each sample taken, and W is the initial total amount of drug in the dialysis bag.

[0036] 5. Experimental Results Figure 7 The median curves represent the cumulative in vitro release behavior of each drug group. The results show that free SA exhibits the fastest release kinetics, with a release rate exceeding 85% within 24 hours and rapidly reaching over 90% in subsequent release processes, without significant inhibition, reflecting its good water solubility. In contrast, free PD has the slowest release process, with a cumulative release rate of less than 70% within 24 hours, and it was not completely released even after 168 hours, with a cumulative release rate of approximately 75%. This release behavior is related to its hydrophobic properties.

[0037] When the two drugs were synthesized into PD-SA NPs, the release of both drugs exhibited controlled-release characteristics: PD showed a stable release mode, with a release rate of about 61% at 24 h, which plateaued after 120 h, with a final release rate of about 88%; SA's release rate was still faster than PD, with a release rate of about 80% at 24 h, which was basically completely released after 120 h, with a final release rate of about 93%, and the final release rate of both drugs was higher than that of the free individual drugs.

[0038] Experiment Example 2: Tyrosinase Inhibition Experiment to Evaluate the In Vitro Whitening Efficacy of PD-SA NPs 1. Experimental Principle Tyrosinase is a key rate-limiting enzyme in the melanin synthesis pathway, specifically catalyzing the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) to the colored product dopachrome, which exhibits a characteristic absorption peak at 475 nm. By measuring the degree of inhibition of this enzymatic reaction in samples, its in vitro skin-whitening activity can be quantitatively evaluated. This experimental example compares the inhibitory activities of PD-SANPs, SA single drugs, and PD-SA NPs nanoparticles on mushroom tyrosinase through three independent experiments, and determines the half-maximal inhibitory concentration (IC50) of PD-SANPs and the positive control kojic acid. 50 ).

[0039] 2. Experimental Preparation Mushroom tyrosinase solution (400 U / mL), L-DOPA solution (14.4 mM), kojic acid standard solution (1 mg / mL), PD solution (1 mg / mL), SA solution (1 mg / mL), PD-SA NPs dispersion (1 mg / mL), PBS buffer, glass cuvettes, UV-Vis spectrophotometer, 37℃ constant temperature water bath, etc.

[0040] 3. Experimental Methods 3.1 Comparison of tyrosinase inhibitory activities of PD, SA, PD-SA NPs and kojic acid: 3.1.1 Experimental grouping: The total reaction volume for all groups was 3 mL, and the specific groupings are as follows: (1) Negative control group: 1.425 mL PBS buffer + 75 μL tyrosinase solution + 1.5 mL L-DOPA solution; (2) PD group: 1.275 mL PBS buffer + 150 μL 1 mg / mL PD solution + 75 μL tyrosinase solution + 1.5 mL L-DOPA solution; (3) SA group: 1.275 mL PBS buffer + 150 μL 1 mg / mL SA solution + 75 μL tyrosinase solution + 1.5 mL L-DOPA solution; (4) Kojic acid group: 1.275 mL PBS buffer + 150 μL 1 mg / mL kojic acid solution + 75 μL tyrosinase solution + 1.5 mL L-DOPA solution; (5) PD-SA NPs group: 1.275mL PBS buffer + 150μL 1mg / mL PD-SA NPs dispersion + 75μL tyrosinase solution + 1.5 mL L-DOPA solution.

[0041] 3.1.2 Experimental Procedure (1) Add the corresponding sample solution and PBS buffer to each group of cuvettes respectively; (2) Add 75 μL of tyrosinase solution (400 U / mL) to all cuvettes, mix gently, and then incubate in a 37°C water bath for 30 min. (3) After the pre-incubation is completed, quickly add 1.5 mL of L-DOPA solution (14.4 mM) preheated to 37°C to all cuvettes, mix well immediately and place in a 37°C constant temperature water bath for 30 min; (4) After the reaction is complete, the absorbance of each cuvette is measured at a wavelength of 475 nm using a spectrophotometer; (5) Calculate the inhibition rate of tyrosinase in each group according to the following formula: Inhibition rate (%) = [1 - (A475)] sample / A475 control )]×100%; A475 sample : Absorbance of the reaction after adding the inhibitor (experimental group); A475 control : Absorbance of the negative control group.

[0042] 3.2 PD-SA NPs half-maximal inhibitory concentration (IC50) 50 Measurement: 3.2.1 Experimental Grouping The total reaction volume for all groups was 3 mL, and six concentration gradients were set up: (1) Negative control group: 1.425 mL PBS buffer + 75 μL tyrosinase solution + 1.5 mL L-DOPA solution; (2) 12.5 μg / mL PD-SA NPs: 1.3875 mL PBS + 37.5 μL 1 mg / mL PD-SA NPs + 75 μL tyrosinase solution + 1.5 mL L-DOPA solution; (3) 25 μg / mL PD-SA NPs: 1.350 mL PBS + 75.0 μL 1 mg / mL PD-SA NPs + 75 μL tyrosinase solution + 1.5 mL L-DOPA solution; (4) 50 μg / mL PD-SA NPs: 1.275 mL PBS + 150.0 μL 1 mg / mL PD-SA NPs + 75 μL tyrosinase solution + 1.5 mL L-DOPA solution; (5) 100 μg / mL PD-SA NPs: 1.125 mL PBS + 300.0 μL 1 mg / mL PD-SA NPs + 75 μL tyrosinase solution + 1.5 mL L-DOPA solution; (6) 200 μg / mL PD-SA NPs: 0.825 mL PBS + 600.0 μL 1 mg / mL PD-SA NPs + 75 μL tyrosinase solution + 1.5 mL L-DOPA solution.

[0043] 3.2.2 Experimental Procedure (1) Add the corresponding sample solution and PBS buffer to each group of cuvettes respectively; (2) Add 75 μL of tyrosinase solution (400 U / mL) to all cuvettes, mix gently, and then incubate in a 37°C water bath for 30 min. (3) After the pre-incubation is completed, quickly add 1.5 mL of L-DOPA solution (14.4 mM) preheated to 37°C to all cuvettes, mix well immediately and start timing; (4) Monitor the absorbance change of each cuvette continuously at a wavelength of 475 nm for 20 min, and record the data once every 1 min; (5) Data calculation and analysis: a. Calculation of initial reaction rate: Plot the reaction kinetic curve with the detection time (min) as the x-axis and the corresponding absorbance value (A475) as the y-axis. Take the slope of the initial linear phase (R²>0.99) in the first 5 min of the curve as the initial rate (ΔA475 / min) of the reaction. b. Calculation of tyrosinase inhibition rate: The inhibition rate of different concentrations of PD-SA NPs on mushroom tyrosinase was calculated according to the following formula: Inhibition rate (%) = [1 - (ΔA475)] sample / ΔA475 control )]×100%, Where ΔA475 control The initial rate of response in the negative control group (no inhibitor), ΔA475 sample : Initial rate of reaction in each inhibitor concentration experimental group.

[0044] c.IC 50 Value fitting: A dose-response curve was plotted with the final concentration of PD-SA NPs on the x-axis and the corresponding tyrosinase inhibition rate on the y-axis. The half-maximal inhibitory concentration (IC50) was calculated using a nonlinear regression model fitted with GraphPad Prism software. 50 ).

[0045] 3.3 Kojic acid half-maximal inhibitory concentration (IC50) 50 Measurement: Using the same 3.0 mL reaction system and experimental procedures as in 3.2, kojic acid solutions with six final concentration gradients of 0, 12.5, 25, 50, 100, and 200 μg / mL were prepared.

[0046] 4. Experimental Results: 4.1 Comparison of tyrosinase inhibitory activities At the same mass concentration of 50 μg / mL, PD-SA NPs exhibited stronger tyrosinase inhibitory activity, and the effect was superior to PD or SA alone. Figure 8 ).

[0047] 4.2 ICs for PD-SA NPs 50 Measurement results Absorbance-time curves under different concentrations of PD-SA NPs ( Figure 9 The results showed that all groups exhibited good linearity (R²>0.99) in the first 5 minutes of the reaction, consistent with first-order kinetics of the enzyme reaction. As the concentration of PD-SA NPs increased, the slope of the curve gradually decreased, indicating that the initial reaction rate of L-DOPA oxidation catalyzed by tyrosinase decreased in a dose-dependent manner with increasing inhibitor concentration. Inhibition rate-concentration dose-response curves of PD-SA NPs were plotted based on the inhibition rates at each concentration. Figure 10 The IC50 of PD-SA NPs against tyrosinase was calculated using nonlinear regression fitting with GraphPad Prism software. 50 The value was 33.4 μg / mL.

[0048] 4.3 IC50 of kojic acid 50 Measurement results Absorbance-time curves under different concentrations of kojic acid ( Figure 11 It also showed a good linear relationship and dose-dependent characteristics. Inhibition rate-concentration dose-response curves of kojic acid were plotted based on the inhibition rate at each concentration. Figure 12 The IC50 of kojic acid on tyrosinase was obtained through fitting calculations. 50 The value was 48.5 μg / mL.

[0049] It can be seen that the IC of PD-SA NPs 50 The levels were significantly lower than those of kojic acid, a traditional tyrosinase inhibitor, which provides important experimental evidence for the application of PD-SA NPs in skin whitening and related fields.

[0050] Experiment Example 3: Evaluation of the in vitro antioxidant efficacy of PD-SA NPs by DPPH free radical scavenging experiment 1. Experimental Principle DPPH (1,1-diphenyl-2-picrylhydrazine) is a commonly used free radical scavenging agent with a purple absorbance. When reacting with antioxidants, the purple color of DPPH gradually fades and the absorbance value decreases, indicating that free radicals are scavenged. By measuring the change in absorbance after the reaction, the scavenging ability of different concentrations of PD-SA NPs against DPPH free radicals can be evaluated, thereby indirectly assessing its antioxidant efficacy.

[0051] 2. Experimental Preparation DPPH solution, anhydrous ethanol, vitamin C solution, PD solution, SA solution, PD-SA NPs dispersion, PBS buffer, 96-well plate, microplate reader, etc.

[0052] 3. Experimental Grouping The reaction was performed using 96-well plates, with the following wells: control group (PBS buffer + DPPH solution), blank control group (PBS buffer only), PD group (PD solution + DPPH solution), blank PD group (PD solution + PBS buffer), SA group (SA solution + DPPH solution), blank SA group (SA solution + PBS buffer), positive control group (vitamin C solution + DPPH solution), blank positive control group (vitamin C solution + PBS buffer), nanoparticle group (PD-SA NPs dispersion + DPPH solution), and blank nanoparticle group (PD-SA NPs dispersion + PBS buffer). Each group had 3 replicates.

[0053] 4. Experimental Procedure (1) Accurately weigh DPPH standard and prepare a 100µM stock solution with anhydrous ethanol; (2) Add 100 µL of DPPH stock solution to each well of all groups containing DPPH solution; add 100 µL of PBS buffer to each well of all groups containing PBS buffer; add only 200 µL of PBS buffer to the control blank group. (3) Add 100 μL of different reagent solutions to each replicate well: 50 μg / mL PD solution to the PD group and PD blank group; 50 μg / mL SA solution to the SA group and SA blank group; 50 μg / mL vitamin C solution to the positive control group and positive control blank group; and 50 μg / mL PD-SA NPs dispersion to the nanoparticle group and nanoparticle blank group. Incubate the 96-well plate in a 37°C incubator in the dark for 30 minutes. (4) After the reaction is complete, the absorbance of each well is measured at 517 nm using an enzyme-linked immunosorbent assay (ELISA) reader. (5) Calculate the DPPH free radical scavenging rate using the following formula: A 517Control =A517 (control) - A517 (control blank) A517 Sample =A517(sample) - A517(sample blank) DPPH free radical scavenging rate (%) = [1 - (A517)] Sample / A517 Control )]×100%.

[0054] 5. Experimental Results The results showed that PD-SA NPs outperformed PD or SA alone in DPPH free radical scavenging. The scavenging rate of PD-SANPs was approximately 80% or higher, and compared to traditional antioxidants such as vitamin C, PD-SA NPs demonstrated superior free radical scavenging ability in in vitro experiments. Figure 13 This indicates that PD-SA NPs have broad application prospects, especially in the prevention of skin aging, anti-aging, and antioxidant-related diseases.

[0055] Experiment Example 4: Evaluation of the in vitro antioxidant efficacy of PD-SA NPs by ABTS free radical scavenging experiment 1. Experimental Principle ABTS (2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) is a commonly used free radical scavenger with a blue-green absorbance. When ABTS reacts with antioxidants, its color fades and its absorbance decreases. By measuring the change in absorbance after the reaction, the scavenging ability of different concentrations of PD-SA NPs against ABTS free radicals can be evaluated, thereby indirectly assessing its antioxidant efficacy.

[0056] 2. Experimental Preparation ABTS solution, potassium persulfate solution, vitamin C solution, PD solution, SA solution, PD-SA NPs dispersion, PBS buffer, 96-well plate, microplate reader, etc.

[0057] 3. Experimental Grouping The reaction was performed using 96-well plates, with the following wells: control group (ABTS + working solution + PBS buffer), control blank group (PBS buffer only), PD group (PD solution + ABTS + working solution), PD blank group (PD solution + PBS buffer), SA group (SA solution + ABTS + working solution), SA blank group (SA solution + PBS buffer), positive control group (vitamin C solution + ABTS + working solution), positive control blank group (vitamin C solution + PBS buffer), nanoparticle group (PD-SA NPs dispersion + ABTS + working solution), and nanoparticle blank group (PD-SA NPs dispersion + PBS buffer). Each group had 3 replicates.

[0058] 4. Experimental Procedure (1) Prepare ABTS reaction solution by mixing 7 mmol / L ABTS stock solution with 2.45 mmol / L potassium persulfate solution in equal volume, react in the dark for 12-16 h, dilute ABTS reaction solution with PBS buffer to make absorbance at 734 nm 0.70±0.02, and finally prepare ABTS+· working solution.

[0059] (2) Add 100 µL of ABTS+ working solution to each well in all groups of plates containing ABTS+ working solution; add 100 µL of PBS buffer to each well in all groups of plates containing PBS buffer; add only 200 µL of PBS buffer to the control blank group. (3) Add 100 μL of different reagent solutions to each replicate well: 50 μg / mL PD solution to the PD group and PD blank group, 50 μg / mL SA solution to the SA group and SA blank group, 50 μg / mL vitamin C solution to the positive control group and positive control blank group, and 50 μg / mL PD-SA NPs dispersion to the nanoparticle group and nanoparticle blank group. Place the 96-well plate in a 37℃ incubator and react in the dark for 30 minutes. (4) After the reaction is complete, the absorbance of each well is measured at 734 nm using an enzyme-linked immunosorbent assay (ELISA) reader. (5) Calculate the ABTS clearance rate using the following formula: A734 Control =A734 (control) - A734 (control blank) A 734 Sample =A734(sample) - A734(sample blank) ABTS clearance rate (%) = [1 - (A 734 Sample / A734 Control )]×100%.

[0060] 5. Experimental Results The results showed that PD-SA NPs outperformed PD or SA alone in ABTS free radical scavenging. PD-SANPs achieved a scavenging rate of over 70%, demonstrating stronger free radical scavenging ability in in vitro experiments compared to traditional antioxidants such as vitamin C. Figure 14 This suggests that they have greater potential for applications in areas related to skin aging or anti-oxidation.

[0061] Based on the comprehensive examples and experimental cases, the polygalactoside-aescin sodium core-shell nanoparticles PD-SA NPs prepared by the present invention have the advantages of stable structure, relatively simple preparation process and scalability, and can meet the application requirements of skin whitening and anti-oxidation, and have good application prospects.

Claims

1. A polydatin- esculoside sodium core-shell nanoparticle, characterized in that, The polydipsia-aescin sodium core-shell nanoparticles are formed by polydipsia as the core and aescin sodium as the shell through an antisolvent precipitation method, and the nanoparticles have a particle size of 50-200 nm.

2. The polygalactoside-aescin sodium core-shell nanoparticles according to claim 1, characterized in that, The mass ratio of polygalactoside to sodium aescinate is 1:1.2-2.

8.

3. The polygalactoside-aescin sodium core-shell nanoparticles according to claim 1 or 2, characterized in that, The mass ratio of polygalactoside to sodium aescinate is 1:1.6-2.

0.

4. The polygalactoside-aescin sodium core-shell nanoparticles according to claim 1 or 2, characterized in that, The mass ratio of polygalactoside to sodium aescinate is 1:1.6-1.

8.

5. The method for preparing polygalactoside-aescin sodium core-shell nanoparticles according to claim 1, characterized in that, Includes the following steps: (1) Dissolve polydipsia glycoside in a mixed solvent of acetone and ethanol to obtain a polydipsia glycoside organic phase solution; (2) Dissolve sodium aescinate in deionized water to obtain an aqueous solution of sodium aescinate; (3) Under high shear stirring conditions, the organic phase solution of Polygonum cuspidatum was added dropwise to the aqueous phase solution of sodium aescinate to form a core-shell nanoparticle dispersion system; (4) The organic solvent was removed by a rotary evaporator under reduced pressure to obtain an aqueous dispersion of polysaccharide-aescin sodium; (5) The above polydipsia-aescin sodium aqueous dispersion was freeze-dried to obtain polydipsia-aescin sodium core-shell nanoparticles.

6. The preparation method according to claim 5, characterized in that, The more specific steps are as follows: S1: Preparation of the organic phase of polydipsia glycoside: Weigh polydipsia glycoside and dissolve it in an organic solvent to obtain a polydipsia glycoside organic phase solution; the organic solvent is a mixed solvent of acetone and ethanol in a volume ratio of 1:1-3, and the mass concentration of the solution is 1-8 mg / mL; S2: Preparation of sodium aescinate aqueous phase: Weigh sodium aescinate, dissolve sodium aescinate in deionized water, and magnetically stir for 30-40 min at 20-35℃ and 800-1000 rpm, followed by ultrasonic treatment for 10-20 min to obtain sodium aescinate aqueous phase solution; the mass concentration of the solution is 1-8 mg / mL. S3: Dropping and High-Shear Dispersion: Place the sodium aescinate aqueous solution in a high-shear mixer container, and set the high-shear mixer speed to 10000-13000 rpm; add the polygalactosyl glycoside organic solution dropwise to the above sodium aescinate aqueous solution at a rate of 1-3 mL / min, and maintain high-shear stirring during the dropwise addition. After the dropwise addition is completed, continue high-shear stirring for 5-15 min to obtain a homogeneous emulsion-dispersion system; S4: Removal of organic solvents: The obtained dispersion system was transferred to a rotary evaporator under reduced pressure and evaporated under reduced pressure in a water bath at 25-45℃ to remove acetone and ethanol, and PD-SA NPs aqueous dispersion was obtained. S5: Purification: The PD-SA NPs aqueous dispersion was separated by centrifugation or ultrafiltration, resuspended in deionized water and washed 2-5 times; finally, the sample was freeze-dried to obtain polydipsia glycoside-aescin sodium core-shell nanoparticles, which were in the form of off-white powder solid.

7. The preparation method according to claim 6, characterized in that, The ultrasonic treatment described in S2 is a frequency of 40kHz and a power of 30%-80%.

8. The application of the polygalactoside-aescin sodium core-shell nanoparticles according to claim 1 in the preparation of skin whitening and antioxidant products.

9. The application according to claim 8, characterized in that, The core-shell nanoparticles achieve skin whitening and antioxidant effects by inhibiting tyrosinase activity and / or scavenging free radicals.

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