Preparation method of cerium-based antioxidant nanoscale enzyme and application thereof in nerve protection medicine

Abstract

The application discloses a preparation method of cerium-based antioxidant nanoscale enzyme and neural protection application, and belongs to the technical field of biological materials. The preparation method comprises the following steps: forming a Ce-based natural product nanoparticle core with an ultrasmall size through coordination in an aqueous phase by using polyvinylpyrrolidone, cerium chloride and a natural product; and then loading astaxanthin through electrostatic adsorption of the CeGA nanoparticle core and the astaxanthin to form a CeGA@ATX nanoscale enzyme. The preparation method has mild conditions and a simple process. The prepared nanoscale enzyme has uniform size, good dispersibility and biocompatibility, and simultaneously has catalase and superoxide dismutase mimetic enzyme activities, and can efficiently remove active oxygen free radicals. In-vitro cell experiments prove that the nanoscale enzyme can significantly improve the survival rate of neuron cells under oxidative stress, reduce the ROS level in cells, and exhibit an inhibiting effect on ferroptosis, and has a wide application prospect in the preparation of medicines or health foods for preventing or treating Parkinson's disease and other neurodegenerative diseases.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological materials, and particularly relates to a preparation method of a cerium-based antioxidant nanoscale enzyme and neural protection application thereof. BACKGROUND

[0002] Oxidative stress is one of the core pathological factors of various neurodegenerative diseases (such as Parkinson's disease, Alzheimer's disease, etc.). Its essence is the imbalance between the generation and clearance of reactive oxygen species (ROS) in the brain, leading to the accumulation of excessive ROS, and then causing lipid peroxidation, protein denaturation, DNA damage and mitochondrial dysfunction, and ultimately inducing neuronal apoptosis or ferroptosis. At present, there is still a lack of means to effectively block or reverse this pathological process in clinical practice, and therefore, it is of great significance to develop an antioxidant that can efficiently and continuously remove ROS.

[0003] Nanoscale enzyme is a kind of nanomaterial with natural enzyme catalytic activity. Compared with natural enzymes, nanoscale enzymes have the advantages of low cost, high stability and large-scale preparation. Among the many nanoscale enzymes, cerium-based nanoscale enzymes exhibit excellent superoxide dismutase (SOD) and catalase (CAT) mimetic activities due to the reversible conversion between Ce³⁺ / Ce 4 ⁺ valence states, and have great potential in the field of antioxidant. However, the catalytic activity center of a single cerium-based nanoscale enzyme is limited, and the antioxidant efficiency in complex biological environments still needs to be improved.

[0004] On the other hand, natural small molecule antioxidants such as gallic acid (GA) and astaxanthin (ATX) have attracted much attention due to their strong free radical scavenging ability. The multiple phenolic hydroxyl groups in the gallic acid molecule can effectively neutralize free radicals; astaxanthin is known as "super vitamin E" due to its unique long conjugated structure, and has extremely strong antioxidant capacity. However, these small molecules face some insurmountable bottlenecks in practical applications: first, poor water solubility, gallic acid has limited solubility in water, and astaxanthin is almost insoluble in water, which seriously limits its distribution and utilization in the body; second, poor stability, both of them, especially astaxanthin, are sensitive to light, heat and oxygen, and are easily degraded and inactivated; third, low bioavailability, the above defects lead to poor oral absorption and rapid metabolism in vivo, making it difficult to reach an effective therapeutic concentration at the lesion site. In the prior art, although some studies have attempted to combine small molecule antioxidants with nanomaterials, there are still some problems: or only simple physical mixing, which cannot form a stable composite structure and has limited effect; or the preparation process is complex and the conditions are harsh, which is difficult to reproduce; or the functional synergy of multiple active ingredients cannot be achieved, and the comprehensive antioxidant performance and protection effect against neurodegenerative diseases are not ideal.

[0005] Therefore, there is an urgent need in the art for a new type of nanomaterial that can integrate the advantages of cerium-based nanoszymes and natural small molecule antioxidants, and overcome their respective defects. The ideal technical solution should be able to: significantly improve the water solubility and stability of small molecules through rational structural design; achieve synergistic enhancement of cerium-based nanoszymes and various small molecule antioxidants in activity, enhance their antioxidant performance; and ultimately prove its clear and efficient neuroprotective effect in complex nervous system diseases. SUMMARY

[0006] The primary purpose of the present application is to overcome the shortcomings of the prior art, to address the problems of poor water solubility, low chemical stability, limited bioavailability, and poor antioxidant effect of single component of natural antioxidants such as astaxanthin and gallic acid, and to provide a new preparation method of cerium-based antioxidant nanoszyme and its application.

[0007] Another purpose of the present application is to provide a natural product nanoszyme with synergistically enhanced antioxidant activity, good water solubility, and high stability prepared by the method, and to expand its application in neuroprotective agents, particularly in the prevention and / or treatment of neurodegenerative diseases such as Parkinson's disease.

[0008] To achieve the above-mentioned purposes, the present application adopts the following technical solutions:

[0009] In a first aspect, the present application provides a preparation method of cerium-based antioxidant nanoszyme, comprising the following steps:

[0010] Step S1: Preparation of cerium-based natural product nanoparticle core. Dissolve polyvinylpyrrolidone (PVP) in water to form a solution, then add an aqueous solution of cerium chloride (CeCl3) and natural products, mix and stir to react, concentrate the reaction solution by ultrafiltration to obtain a Ce-based natural product nanoparticle dispersion.

[0011] Step S2: Preparation of cerium-based nanoszyme. Dissolve astaxanthin (ATX) in dimethyl sulfoxide (DMSO) to prepare an astaxanthin solution; mix and stir the Ce-based natural product nanoparticle dispersion of step S1 with the astaxanthin solution, remove DMSO by dialysis after reaction, remove the precipitate by centrifugation, collect the supernatant and concentrate by ultrafiltration to obtain the cerium-based antioxidant nanoszyme solution.

[0012] Further, the molar ratio of CeCl3 to polyphenol and flavonoid natural products in step S1 is 2:1-5:1.

[0013] Further, the concentration of PVP (30kDa) prepared in step S1 is 10-30 mg / mL.

[0014] Further, the concentration of the polyphenol and flavonoid natural product solution in step S1 is 5-10 mg / mL. The natural product is a polyphenol and flavonoid compound, including gallic acid, quercetin, and epigallocatechin gallate, etc.

[0015] Further, the stirring reaction time in step S1 is 48-72 h, the ultrafiltration tube used is 100KD, and the ultrafiltration condition is 4500 rpm centrifugation for 10 min each time.

[0016] Further, the concentration of the ATX in step S2 is 3-8 mg / mL.

[0017] Further, the volume ratio of the Ce-based natural product nanoparticle solution to the ATX solution in step S2 is 1:3-1:6.

[0018] Further, the stirring in step S2 is in the dark at 4°C, and the room temperature reaction time is 48-72 h.

[0019] Further, the dialysis bag used in step S2 is 8K-14K, and the dialysis time is 24 h.

[0020] In a second aspect, the present application provides a cerium-based antioxidant nanoscale enzyme prepared by the method of the first aspect.

[0021] Preferably, the core size distribution of the nanoscale enzyme is 1-5 nm.

[0022] Preferably, the average hydrated particle size of the nanoscale enzyme is about 180-260 nm.

[0023] Preferably, the nanoscale enzyme has SOD mimetic enzyme and POD mimetic enzyme activities.

[0024] The antioxidant performance of the nanoscale enzyme can be determined by an enzyme-like activity detection kit, and the ROS scavenging capacity is characterized by a DCFH-DA fluorescent probe and an ABTS free radical (ABTS· + ) scavenging method.

[0025] In a third aspect, the present application provides a pharmaceutical combination, characterized in that it comprises the cerium-based antioxidant nanoscale enzyme of the second aspect and a pharmaceutically acceptable carrier.

[0026] In a fourth aspect, the present application provides the use of the cerium-based antioxidant nanoscale enzyme of the second aspect or the pharmaceutical combination of the third aspect in the preparation of a product for the following purposes:

[0027] a) a health food or a drug for antioxidant and / or neuroprotection;

[0028] b) a drug for preventing and / or treating diseases caused by oxidative stress;

[0029] c) a drug for preventing and / or treating Parkinson's disease or other neurodegenerative diseases.

[0030] Preferably, in the application, the nano-enzyme or drug combination exerts a neuroprotective effect through one or more of the following mechanisms:

[0031] (1) increasing the survival rate of neurons under oxidative stress conditions;

[0032] (2) reducing the level of ROS in cells;

[0033] (3) regulating iron metabolism in cells;

[0034] (4) inhibiting neuronal ferroptosis.

[0035] Beneficial effects

[0036] Compared with the prior art, the technical solution provided by the present application has the following advantages:

[0037] (1) The present application forms a CeGA nano-enzyme core through the coordination of cerium ions and natural products, and further efficiently loads astaxanthin through intermolecular forces, constructing an inorganic-organic composite nano-enzyme system. This structure realizes the activity synergy of cerium-based nano-enzyme and two kinds of natural antioxidants, and its antioxidant performance, free radical scavenging ability, and SOD and POD double enzyme simulation activity are significantly better than those of single cerium-based nanoparticles.

[0038] (2) The present application successfully loads the hydrophobic astaxanthin in the hydrophilic nanoparticles, greatly improving the water solubility and dispersion stability of astaxanthin in the system, and effectively enhancing the chemical stability of natural products in solution, effectively preventing their rapid degradation and inactivation, thereby greatly improving the bioavailability of active ingredients.

[0039] (3) In vitro cell experiments confirm that the CeGA@ATX nano-enzyme of the present application can effectively alleviate the oxidative stress damage of neurons induced by MPP + , reduce the excessive ROS level in cells, and significantly improve the survival rate of neuronal cells. It can also regulate iron metabolism in cells, inhibit ferroptosis, and exert a neuroprotective effect through multiple pathways, providing a new and effective way for the prevention and treatment of Parkinson's disease and other neurodegenerative diseases. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1The images show the physical appearance of the three cerium-based natural product nanoparticles (CeGA, CeQe, CeEGCG) prepared in Example 1 of this invention, visually demonstrating their aqueous dispersibility and solution state.

[0041] Figure 2 The image (a) shows the transmission electron microscope (TEM) image of the CeGA nanoparticles prepared in Example 1 of this invention, and its hydrated particle size distribution (b).

[0042] Figure 3 The image shows a TEM image (a) and a hydrated particle size distribution diagram (b) of the CeQe nanoparticles prepared in Example 1 of this invention.

[0043] Figure 4 The image shows a TEM image (a) and a hydrated particle size distribution diagram (b) of the CeEGCG nanoparticles prepared in Example 1 of this invention.

[0044] Figure 5 The images show the TEM morphology (a) and size distribution statistics (b) of CeGA prepared in Example 2 of this invention, and the TEM morphology (c) and size distribution statistics (d) of CeGA@ATX nanozyme.

[0045] Figure 6 The image shows a comparison of the hydrated particle size of CeGA and CeGA@ATX nanozymes prepared in Example 2 of this invention (a) and their dot plot (b).

[0046] Figure 7 The UV-Vis absorption spectra of CeGA and CeGA@ATX nanozymes in Example 3 of this invention are shown.

[0047] Figure 8 The results of ABTS radical scavenging rate determination of CeGA and CeGA@ATX nanozymes in Example 4 of the present invention are shown in (a) and (b) respectively.

[0048] Figure 9 The results of the POD mimic enzyme activity test (a) and SOD mimic enzyme activity test (b) of CeGA and CeGA@ATX in Example 4 of the present invention are shown.

[0049] Figure 10 In Example 5 of this invention, the effects of different concentrations of CeGA and CeGA@ATX nanozymes on the survival rate of SH-SY5Y cells were determined using the CCK-8 assay.

[0050] Figure 11 In Example 5 of this invention, CeGA@ATX nanozyme targets the neurotoxin MPP. +Protective effect of induced SH-SY5Y cell injury model.

[0051] Figure 12 This is a fluorescence detection image of the reactive oxygen species level in SH-SY5Y cells under different treatment conditions (DCFH-DA probe method) in Example 6 of the present invention, used to evaluate the ROS scavenging effect of CeGA and CeGA@ATX nanozymes in a Parkinson's cell model.

[0052] Figure 13 This is the result of the test on the enhancement of the total antioxidant capacity of SH-SY5Y cells using CeGA@ATX nanozyme in Example 6 of the present invention.

[0053] Figure 14 In Example 6 of this invention, CeGA@ATX nanozyme targets MPP. + The effect of glutathione levels in a damaged cell model.

[0054] Figure 15 The image (a) and its quantitative analysis (b) of mitochondrial morphology and function shown by Mito Tracker Red staining in Example 7 of this invention are used to evaluate the protective effect of CeGA@ATX nanozymes on mitochondria.

[0055] Figure 16 In Example 7 of this invention, immunofluorescence staining (a) and quantitative analysis of fluorescence intensity of tyrosine hydroxylase TH in SH-SY5Y cells of different treatment groups were used to evaluate the protective effect of CeGA@ATX on dopaminergic neuronal markers.

[0056] Figure 17 In Example 7 of this invention, immunofluorescence staining (a) and quantitative fluorescence analysis (b) of intracellular synaptophysin (SYP) in SH-SY5Y cells of different treatment groups were used to evaluate the ability of CeGA@ATX nanozymes to improve neuronal function in a Parkinson's disease model.

[0057] Figure 18 The Western Blot results (a) and quantitative analysis (bd) of proteins related to iron metabolism and ferroptosis in Example 8 of the present invention are shown. Detailed Implementation

[0058] Example 1

[0059] This embodiment provides a method for preparing Ce-based natural product nanoparticles (CeGA, CeQe, CeEGCG):

[0060] 600 mg of polyvinylpyrrolidone (PVP, Mw = 30 kDa) was accurately weighed and dissolved in 20 mL of ultrapure water. The solution was magnetically stirred until completely dissolved, forming a clear PVP solution. Subsequently, 81 mg of cerium chloride (CeCl3) powder and 2 mL of a 10 mg / mL aqueous solution of natural products (gallic acid (GA), quercetin (Qe), epigallocatechin gallate (EGCG), etc.) were added sequentially to the PVP aqueous solution. The mixture was stirred at room temperature for 72 h. After the reaction was completed, the reaction solution was centrifuged at 10,000 rpm for 10 min, and the supernatant was collected. The supernatant was concentrated by ultrafiltration using a 100 kDa ultrafiltration tube at a centrifugal force of 4500 × g to remove unreacted small molecules and solvent. The concentrated solution was collected to obtain a dispersion of cerium-based natural product nanoparticles (CeGA, CeQe, or CeEGCG), which was stored at 4 °C for later use.

[0061] The prepared cerium-based natural product nanoparticles all exhibit excellent water solubility and dispersibility, and their solutions are clear and transparent. Figure 1 Transmission electron microscopy results showed that the obtained nanoparticles had regular morphology and uniform size (range of 1-5 nm), with a hydrated particle size distribution of approximately 9 nm. The results are as follows: Figures 2-4 As shown.

[0062] Example 2

[0063] This embodiment provides a method for preparing CeGA@ATX nanozymes:

[0064] Step 1 is performed in the same manner as in Example 1, wherein the natural product is gallic acid (GA), and CeGA nanoparticle dispersion is finally obtained.

[0065] Step 2: Synthesis of CeGA@ATX nanozyme: Astaxanthin (ATX) was accurately weighed and dissolved in dimethyl sulfoxide (DMSO), with ultrasonication to aid dissolution, to prepare an ATX stock solution with a concentration of 4 mg / mL. The CeGA nanoparticle dispersion obtained in Step 1 was mixed with the above ATX solution at a volume ratio of 1:4. The mixture was placed at 4°C and magnetically stirred in the dark for 72 h to ensure sufficient astaxanthin loading. After the reaction was complete, the mixture was transferred to a dialysis bag with a molecular weight cutoff of 8-14 kDa and dialyzed in ultrapure water for 24 h to completely remove DMSO. The dialyzed solution was centrifuged (12,000 rpm, 10 min) to remove trace precipitates. The supernatant was collected and concentrated by ultrafiltration to obtain the final CeGA@ATX nanozyme solution, which was stored at 4°C in the dark.

[0066] Example 3

[0067] The properties of the CeGA nanoparticles prepared in Example 1 and the CeGA@ATX nanozymes prepared in Example 2 were characterized as follows:

[0068] (1) Morphology and size observation. The morphology of the nanozyme was observed using a transmission electron microscope (TEM, model FL-200). The results showed that ( Figure 5 CeGA@ATX nanozymes are slightly larger than CeGA nanoparticles, approximately (2.9±0.26) nm.

[0069] (2) Analysis of hydrated particle size and zeta potential. For example... Figure 5 As shown, the average hydrated particle size of CeGA@ATX is approximately 180-260 nm. Zeta potential results indicate that ( Figure 6 CeGA nanoparticles have a negative charge (approximately -3mV) on their surface, while astaxanthin is electropositive in solution. The CeGA@ATX nanozyme formed by their combination has a reduced absolute value of the Zeta potential, further confirming the combination of the two.

[0070] (3) Ultraviolet-Vis Spectroscopy Analysis. Ultraviolet-visible spectral scanning was performed on CeGA nanoparticles and CeGA@ATX nanozyme solution. For example... Figure 7 As shown, the CeGA@ATX nanozyme exhibits a characteristic absorption peak of astaxanthin around 470 nm, while GA and Ce are present at 270 nm. 3+ The characteristic absorption of the coordination structure confirms that CeGA nanoparticles were successfully loaded with ATX.

[0071] Example 4

[0072] The in vitro antioxidant capacity of the CeGA nanoparticles and CeGA@ATX nanozymes prepared in Example 2 was determined:

[0073] (1) ABTS free radical scavenging activity. The total antioxidant capacity assay kit (ABTS method) was used for evaluation. 20 μL of CeGA or CeGA@ATX nanozyme solution of the same concentration was mixed with 180 μL of pre-activated ABTS working solution, and the mixture was reacted at room temperature in the dark for 5 min. The absorbance was measured at 734 nm. Figure 8 As shown, both CeGA and CeGA@ATX nanozymes exhibit concentration-dependent ABTS radical scavenging capabilities, with scavenging rates reaching 79% and 92%, respectively, at the highest tested concentration. Furthermore, CeGA@ATX nanozymes demonstrate superior antioxidant capacity.

[0074] (2) Assay of simulated enzyme activity. The enzyme-like activity of the nanozymes was determined using peroxidase (POD) and superoxide dismutase (SOD) assay kits, strictly following the instructions. Results are as follows: Figure 9As shown, both CeGA and CeGA@ATX exhibited concentration-dependent POD and SOD mimicry activities. At the same concentration, the POD and SOD mimicry activities of CeGA@ATX were significantly higher than those of CeGA nanoparticles, demonstrating that astaxanthin and CeGA nanoparticles had a synergistic effect, with astaxanthin primarily enhancing SOD mimicry activity.

[0075] Example 5

[0076] Cell proliferation-toxicity assays were performed on the CeGA@ATX nanozyme prepared in Example 2:

[0077] (1) Cell compatibility. The cytotoxicity of nanozymes was detected using the CCK-8 assay. SH-SY5Y cells were inoculated at 1×10⁻⁶ cells / year. 5 The cells were seeded at a density of [cells / mL] in 96-well plates and incubated at 37°C with 5% CO2 for 24 h. The medium was then replaced with fresh medium containing different concentrations of CeGA or CeGA@ATX nanozymes, and incubated for another 24 h. The old medium was then aspirated, and CCK-8 reagent diluted with the medium was added and incubated for 3 h. The absorbance at 450 nm was then measured. Figure 10 As shown, at the tested concentration, the cell survival rate of each group was higher than 95%. Under the same concentration conditions, CeGA@ATX nanozymes showed better biocompatibility than CeGA nanoparticles.

[0078] (2) For MPP + Protective effect against induced neuronal damage. Establishment of MPP. + A Parkinson's disease model induced by SH-SY5Y cells. After cell seeding, cells were pretreated with different concentrations of CeGA@ATX nanozymes for 4 h, followed by the addition of cells containing 1 mM MPP. + The cells were cultured in the same medium for another 24 hours, and cell viability was assessed using the CCK-8 assay. Figure 11 As shown, MPP + Pretreatment with CeGA@ATX nanozymes can lead to a significant decrease in cell survival, while pretreatment with CeGA@ATX nanozymes can increase the survival rate of Parkinson's disease model cells in a concentration-dependent manner, demonstrating a significant neuroprotective effect.

[0079] Example 6

[0080] The intracellular antioxidant capacity of the CeGA@ATX nanozyme prepared in Example 2 was determined:

[0081] (1) Intracellular ROS clearance. Intracellular ROS levels were detected using the DCFH-DA fluorescent probe. The experiment was divided into a control group and an MPP group. + Model group, MPP + Combined CeGA nanoparticles and MPP+ Combined CeGA@ATX nanozyme treatment group. Results are as follows: Figure 12 As shown, compared with the control group, MPP + The significantly enhanced green fluorescence intensity in the model group cells indicated elevated ROS levels; while cells pretreated with CeGA and CeGA@ATX showed a significant decrease in intracellular fluorescence intensity, with the CeGA@ATX group showing the best effect, demonstrating its effective ability to scavenge MPP. + Excessive ROS induced.

[0082] (2) Inhibition of lipid peroxidation. Intracellular lipid peroxidation levels were detected using the BODIPY™ 581 / 591C11 fluorescent probe. This probe emits green fluorescence at sites of lipid peroxidation and red fluorescence when lipids are not oxidized. Results are as follows: Figure 13 As shown, MPP + The model group cells exhibited strong green fluorescence, while pretreatment with CeGA@ATX nanozyme significantly inhibited the enhancement of this green fluorescence, demonstrating that it can effectively alleviate lipid peroxidation damage in neuronal cells.

[0083] (3) Restore intracellular glutathione (GSH) levels. Intracellular GSH levels were measured using a commercially available GSH assay kit. For example... Figure 14 As shown, MPP + The intracellular GSH content in the model group cells was significantly reduced, while CeGA@ATX pretreatment could effectively reverse this trend and restore GSH levels to near normal levels, indicating that it can play a protective role by maintaining the intracellular antioxidant system.

[0084] Example 7

[0085] Detection of neuronal cell protection effect of the CeGA@ATX nanozyme prepared in Example 2:

[0086] (1) Mitochondrial protection. Mitochondria were stained with Mito Tracker Red CMXRos fluorescent dye. Observation under confocal microscopy revealed that MPP + Treatment resulted in a significant decrease in mitochondrial red fluorescence in SH-SY5Y cells. Figure 15 This indicates that the mitochondrial membrane potential is impaired; however, the mitochondrial fluorescence intensity of the CeGA@ATX nanozyme pretreated group was significantly stronger, indicating that the nanozyme can effectively protect neurons from MPP. + Induced mitochondrial damage.

[0087] (2) Neuronal function recovery. The effect of CeGA@ATX nanozyme on the expression of neuronal functional proteins was detected by immunofluorescence. Figure 16 and 17The results showed that CeGA@ATX nanozymes could promote MPP + The induced expression levels of tyrosine hydroxylase TH and synaptophysin (SYP), hallmark proteins of dopaminergic neurons, were restored in SH-SY5Y cells. This demonstrates that CeGA@ATX nanozymes can protect neurons from the neurotoxin MPP. + The damage caused can be repaired and neuronal function can be restored.

[0088] Example 8

[0089] Investigation into the neuronal cell protection mechanism of the CeGA@ATX nanozyme prepared in Example 2:

[0090] The expression of intracellular iron metabolism-related proteins was detected using Western blotting. Figure 18 The results showed that MPP + In an induced Parkinson's disease cell model, the expression of iron transporter FPN1, ferritin, and glutathione peroxidase 4 (GPX4) was significantly downregulated, revealing the presence of MPP. + It induces ferroptosis in neurons. However, treatment with CeGA@ATX nanozymes can effectively reverse this trend, restoring the levels of FPN1, Ferritin, and GPX4, demonstrating that it can exert neuroprotective effects by regulating iron metabolism and inhibiting ferroptosis pathways.

Claims

1. A method for preparing a cerium-based antioxidant nanozyme, characterized in that, Includes the following steps: Step S1: Dissolve polyvinylpyrrolidone (PVP) in water to form a solution, then add cerium chloride (CeCl3) and an aqueous solution of the natural product, mix and stir to react, and then concentrate the reaction solution by centrifugation and ultrafiltration to obtain a Ce-based natural product nanoparticle dispersion, wherein the natural product is gallic acid. Step S2: Dissolve astaxanthin ATX in dimethyl sulfoxide (DMSO) to prepare an astaxanthin solution; mix and stir the Ce-based natural product nanoparticle dispersion from step S1 with the astaxanthin solution, remove DMSO by dialysis after reaction, remove the precipitate by centrifugation, collect the supernatant and concentrate it by ultrafiltration to obtain the cerium-based antioxidant nanoenzyme solution.

2. The preparation method according to claim 1, characterized in that, In step S1, the molar ratio of cerium chloride (CeCl3) to the natural product is 2:1-5:

1.

3. The preparation method according to claim 1, characterized in that, The stirring reaction described in step S1 is carried out at room temperature for 48-72 h; the stirring reaction described in step S2 is carried out at 4°C for 48-72 h in the dark.

4. The preparation method according to claim 1, characterized in that, In step S1, the molecular weight of the PVP is 30 kDa, and its aqueous solution concentration is 10-30 mg / mL; the aqueous solution of the natural product is 5-10 mg / mL.

5. The cerium-based antioxidant nanozyme prepared by the method according to any one of claims 1-4, characterized in that, The core size of the cerium-based natural product nanoparticles is 1-5 nm.

6. The cerium-based antioxidant nanozyme according to claim 5, characterized in that, When the natural product is gallic acid, CeGA@ATX nanozyme is obtained, and the size of CeGA@ATX nanozyme is (2.9±0.26) nm.

7. The cerium-based antioxidant nanozyme according to claim 6, characterized in that, Cerium-based antioxidant nanozymes possess superoxide dismutase and catalase mimicry activities.

8. The cerium-based antioxidant nanozyme according to claim 7, characterized in that, The nanozyme exerts its neuroprotective effect through one or more of the following mechanisms: (1) Improve the survival rate of neurons under oxidative stress conditions; (2) Reduce ROS levels in cells; (3) Regulates intracellular iron metabolism; (4) Inhibit neuronal ferroptosis.

9. The use of the cerium-based antioxidant nanozyme according to any one of claims 5 to 8 in the preparation of a drug for preventing Parkinson's disease.

10. The application according to claim 9, which plays a neuroprotective role in the prevention of Parkinson's disease.

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