Copper ion / gallic acid nano-enzyme with superoxide dismutase activity as well as preparation method and application of copper ion / gallic acid nano-enzyme

Abstract

The invention relates to a copper ion / gallic acid nano-enzyme with superoxide dismutase activity as well as a preparation method and application of the copper ion / gallic acid nano-enzyme. The nano-enzyme is formed by reaction of copper ions, gallic acid and sodium hydroxide in deionized water, the reaction is carried out for 1-3 hours under a water bath condition of about 30 DEG C, and the obtained nano-enzyme is obtained by centrifugal enrichment. The molar feeding ratio of the copper ions to the gallic acid to the sodium hydroxide is 1: 1: 0.5 to 1: 1: 2, and the copper source can be copper nitrate trihydrate or copper chloride dihydrate. The nano-enzyme has good dispersity and stability, shows excellent superoxide anion scavenging capacity, simulates the activity of natural superoxide dismutase, is suitable for preparing drugs for treating oxidative stress related diseases, and particularly has wide application prospects in the fields of inflammatory diseases, cardiovascular and cerebrovascular diseases, neurodegenerative diseases and the like.

Description

Technical Field

[0001] This invention relates to the fields of nanomaterials and biomimetic enzyme technology, and in particular to a copper ion / gallic acid nanozyme with superoxide dismutase activity, its preparation method and application. Background Technology

[0002] In research on the treatment of oxidative stress-related diseases, abnormally elevated levels of reactive oxygen species (ROS) are considered a significant factor in inducing various pathological conditions. Numerous studies have shown that ROS accumulation is closely associated with over 200 diseases, including inflammatory diseases such as periodontitis and glomerulonephritis; cardiovascular and cerebrovascular diseases such as ischemia-reperfusion injury and atherosclerosis; and neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. The development mechanisms of these diseases are largely related to cellular oxidative damage; therefore, effectively scavenging ROS has become a crucial strategy for disease intervention.

[0003] Current clinical treatments primarily rely on surgery, physical interventions, and small-molecule antioxidants. However, these traditional methods generally suffer from problems such as high invasiveness, limited efficacy, poor targeting, low bioavailability, and significant side effects, severely restricting their widespread application. Against this backdrop, the rise of nanomedicine technology, especially nanozymes, offers a new direction for the treatment of oxidative stress-related diseases.

[0004] Nanozymes are a class of nanomaterials with functions similar to natural enzymes. Some nanozymes are designed to mimic the activity of superoxide dismutase (SOD), thereby scavenging superoxide anions and inhibiting oxidative damage. In the field of antioxidants, SOD-active nanozymes have become an important research target for replacing natural enzymes due to their advantages such as simple synthesis process, high stability, low cost, and easy storage.

[0005] However, most reported nanozymes still face numerous technical bottlenecks. For example, the lack of targeted design of the catalytic center results in significantly lower catalytic efficiency compared to natural superoxide dismutases (SODs); simultaneously, the potential toxicity of some nanozyme raw materials increases the difficulty of clinical translation. Therefore, developing a nanozyme material that combines high SOD activity, good stability, and biosafety has become one of the key research challenges. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a copper ion / gallic acid nanozyme with superoxide dismutase activity, its preparation method and application, so as to improve the superoxide dismutase activity of the nanozyme, reduce its biotoxicity, and thus have higher practical value and biosafety in the treatment of oxidative stress-related diseases.

[0007] To achieve the above objectives, the present invention provides the following technical solution: In one embodiment of the present invention, a copper ion / gallic acid nanozyme with superoxide dismutase activity is provided. The nanozyme is formed by the reaction of copper ions, gallic acid and base, wherein the molar ratio of copper ions, gallic acid and base is 1:1:0.5 to 1:1:2. The nanozyme has the function of scavenging superoxide anions.

[0008] Furthermore, the alkali is sodium hydroxide.

[0009] In another embodiment of the present invention, a method for preparing copper ion / gallic acid nanozyme is provided, comprising the following steps: dissolving gallic acid and alkali in deionized water and stirring to dissolve; then adding water-soluble copper salt and continuing to stir and mix; then reacting at 30°C for 1-3 hours; finally, centrifuging after the reaction is completed and collecting the precipitate to obtain copper ion / gallic acid nanozyme.

[0010] Preferably, the water-soluble copper salt is copper nitrate trihydrate or copper chloride dihydrate.

[0011] Furthermore, the centrifugation conditions are 10,000 rpm for 10 minutes.

[0012] In one embodiment of the present invention, the copper ion / gallic acid nanozyme can be used to prepare a drug for treating oxidative stress-related diseases.

[0013] Alternatively, the oxidative stress-related diseases may include inflammatory diseases, cardiovascular diseases, or neurodegenerative diseases.

[0014] Preferably, the inflammatory disease is periodontitis or glomerulonephritis; the cardiovascular disease is ischemia-reperfusion injury or atherosclerosis; and the neurodegenerative disease is Alzheimer's disease or Parkinson's disease.

[0015] In another embodiment of the invention, the copper ion / gallic acid nanozyme can also be used to prepare a formulation for scavenging superoxide anions.

[0016] Furthermore, a pharmaceutical composition is provided comprising a copper ion / gallic acid nanozyme and a pharmaceutically acceptable carrier.

[0017] Based on the above technical solution, the copper ion / gallic acid nanozyme with superoxide dismutase activity of the present invention forms a stable nanostructure through the reaction of copper ions, gallic acid, and sodium hydroxide. Optimization of enzyme activity is achieved by utilizing a reasonable molar ratio (1:1:0.5 to 1:1:2) and controlled water bath reaction conditions (30℃, 1-3 hours). Compared with traditional natural superoxide dismutase, this nanozyme not only has a simpler preparation process and lower cost, but also has significant advantages in structural stability, biocompatibility, batch preparation, and preservation.

[0018] This nanozyme exhibits excellent superoxide anion scavenging ability. Its SOD-mimicking activity was verified by WST-8 assay and electron paramagnetic resonance technology, demonstrating significant free radical scavenging efficiency, effectively alleviating oxidative stress levels, and reducing cell and tissue damage. By adjusting the feed ratio and reaction conditions, products with different catalytic capabilities can be obtained, exhibiting good controllability and reproducibility.

[0019] Furthermore, the raw materials for the nanozyme of this invention are widely available, have low toxicity and side effects, and meet the safety requirements for biomedical applications. It exhibits good effects in anti-inflammation, neuroprotection, and maintenance of vascular endothelial function, and is particularly suitable for the treatment of various diseases involving oxidative stress in their pathogenesis, such as periodontitis, glomerulonephritis, ischemia-reperfusion injury, atherosclerosis, Alzheimer's disease, and Parkinson's disease.

[0020] This invention can be applied not only to the field of drug development, such as the preparation of drug compositions or sustained-release systems for scavenging superoxide anions, but also to multiple industries such as cosmetics, food additives, and in vitro antioxidant detection, and has broad market prospects and important socio-economic value. Attached Figure Description

[0021] Figure 1 The image shown is a transmission electron microscope (TEM) image of the copper ion / gallic acid nanozyme prepared in Example 1, used to show the particle size distribution and morphological characteristics of the obtained nanozyme.

[0022] Figure 2 The image shown is a transmission electron microscope image of the copper ion / gallic acid nanozyme prepared in Example 2, used to compare the morphological changes of the nanozyme under different sodium hydroxide dosages.

[0023] Figure 3 The image shown is a transmission electron microscope image of the copper ion / gallic acid nanozyme prepared in Example 3, which further demonstrates the structural morphology of the product particles under high alkali concentration.

[0024] Figure 4The images shown are transmission electron microscope images of copper ion / gallic acid nanozymes prepared using different copper salts (i.e., copper chloride dihydrate) in Example 4, used to compare the effects of different copper sources on the morphology of the nanozymes.

[0025] Figure 5 The image shows a transmission electron microscope image of the nanozyme prepared in Example 5 with a shortened reaction time of 1 hour, reflecting the effect of reaction time on particle formation.

[0026] Figure 6 The image shown is a transmission electron microscope image of the nanozyme prepared in Example 6 with a reaction time of 2 hours, used to further compare the regulatory effect of reaction time on particle structure.

[0027] Figure 7 The graph shows the experimental results of determining the superoxide dismutase activity of the nanozyme prepared in Example 1 by the WST-8 colorimetric method, which is used to illustrate the scavenging ability of the nanozyme for superoxide anions.

[0028] Figure 8 To determine the free radical scavenging ability spectrum of the nanozyme prepared in Example 1 using an electron paramagnetic resonance (ESR) instrument, to demonstrate its inhibitory effect on superoxide anions, and to further prove that it has superoxide dismutase activity. Detailed Implementation

[0029] This invention provides a detailed description of the preparation method, structural characteristics, and enzyme activity of copper ion / gallic acid nanozymes with superoxide dismutase activity through the following examples. These examples are intended to illustrate the technical solutions of this invention and are not intended to limit the scope of protection of this invention.

[0030] Example 1: Preparation and Morphology Analysis of Nanozymes under Standard Feed Ratio This embodiment aims to demonstrate the preparation process and structural morphology characteristics of copper ion / gallic acid nanozymes with superoxide dismutase (SOD) activity synthesized using copper nitrate trihydrate, gallic acid, and sodium hydroxide under standard feeding conditions.

[0031] First, weigh 340.2 mg of gallic acid and 40 mg of sodium hydroxide, add them to 200 mL of deionized water, and stir thoroughly with a magnetic stirrer at room temperature until completely dissolved. This step ensures that gallic acid reacts with sodium hydroxide to form a complex ligand, providing a stable environment for subsequent metal ion complexation.

[0032] Subsequently, 483.2 mg of copper nitrate trihydrate was slowly added to the above transparent solution, and the mixture was stirred for 5 minutes to promote uniform mixing of the reactants. Next, the reaction system was transferred to a water bath at a set temperature of 30°C, and heated and stirred for 3 hours to promote the complexation of copper ions with gallic acid to form a stable nanostructure.

[0033] After the reaction is complete, the reaction solution is removed and immediately centrifuged at 10,000 rpm for 10 minutes. The supernatant is removed, and the lower reddish-brown precipitate is collected, which is the copper ion / gallic acid nanozyme concentrate. The obtained product does not require additional drying treatment and can be directly used for subsequent activity determination and component analysis.

[0034] To verify the particle size and morphology of the prepared nanozymes, the above concentrated sample was analyzed by transmission electron microscopy (TEM). Figure 1 The nanozyme was found to be approximately spindle-shaped particles with a long axis size between 100-120 nm and a short axis size between 20-40 nm. It also showed good overall dispersion with no obvious agglomeration.

[0035] The results indicate that, under the condition of a molar ratio of copper ions: gallic acid: sodium hydroxide = 1:1:1, combined with appropriate reaction temperature and time, copper ion / gallic acid nanozymes with stable structure and good dispersion can be prepared efficiently, providing a physical basis for subsequent enzyme activity evaluation and application development.

[0036] Example 2: Structural regulation under low alkalinity conditions To investigate the effect of alkali dosage on the morphology of copper ion / gallic acid nanozymes, this example reduces the amount of sodium hydroxide added while keeping other reaction conditions constant, and studies its regulatory effect on the nanozyme structure.

[0037] The specific steps are as follows: Weigh 340.2 mg of gallic acid and 20 mg of sodium hydroxide and add them to 200 mL of deionized water. Stir thoroughly at room temperature to ensure that the gallic acid and base in the system can be completely dissolved to form a complex precursor solution.

[0038] Subsequently, 483.2 mg of copper nitrate trihydrate was added, and stirring was continued for 5 minutes to allow the copper ions to fully react with the organic ligands in the precursor solution. After mixing was complete, the entire reaction system was transferred to a water bath at a set temperature of 30°C and the reaction was maintained at a constant temperature with stirring for 3 hours to promote the formation of nanozymes.

[0039] After the reaction was complete, the reaction solution was removed and centrifuged at 10,000 rpm for 10 minutes. The supernatant was discarded after centrifugation, and the resulting precipitate was collected as the nanozyme concentrate.

[0040] The obtained samples were analyzed using transmission electron microscopy (TEM) to observe the microstructure of the nanozymes. Figure 2 The image shown is a TEM image of the product of Example 2. It shows that the particle distribution is not as uniform as that of Example 1, some particles are agglomerated, the particle size uniformity is reduced, and the dispersibility is reduced.

[0041] Analysis suggests that lower alkali concentrations may affect the complexation efficiency of copper ions with gallic acid and the pH stability of the solution, leading to a slower nucleation rate and consequently affecting the particle size uniformity and morphological stability of the product.

[0042] In summary, controlling the appropriate amount of alkali plays a crucial role in the microstructure control of nanozymes. Although nanozymes can be successfully prepared under low alkali conditions, their particle size distribution and morphological stability are slightly inferior to those under standard conditions, and further optimization is needed to meet specific application requirements.

[0043] Example 3: Product Analysis under High Alkalinity Conditions To further investigate the effect of alkali dosage on the structure and morphology of copper ion / gallic acid nanozymes, this embodiment increases the amount of sodium hydroxide on the basis of standard conditions to prepare nanozymes under enhanced alkali concentration conditions, and analyzes their structural characteristics.

[0044] The specific steps are as follows: Weigh 340.2 mg of gallic acid and 80 mg of sodium hydroxide and add them to 200 mL of deionized water. Stir thoroughly with a magnetic stirrer until dissolved, generating a uniform, dark green precursor solution. This relatively high concentration of alkaline environment helps promote the dissociation of gallic acid and enhances its complexing ability with metal ions.

[0045] Subsequently, 483.2 mg of copper nitrate trihydrate was added, and stirring was continued for 5 minutes to ensure that the copper ions were fully and uniformly mixed with the complexed ligands formed in the reaction system. Then, the mixed solution was transferred to a 30°C water bath and heated and stirred continuously for 3 hours.

[0046] After the reaction is complete, the reaction solution is centrifuged at 10,000 rpm for 10 minutes, the supernatant is discarded, and the lower precipitate is collected, which is the copper ion / gallic acid nanozyme product prepared under high alkaline conditions.

[0047] The prepared nanozyme samples were sent to a transmission electron microscope (TEM) for morphological observation. Figure 3The TEM images shown indicate that the spindle-shaped nanoenzyme particles formed under high-alkali conditions have smaller long axis dimensions and larger short axis dimensions, are more uniformly distributed, exhibit a good spindle-shaped structure, and show no obvious aggregation.

[0048] Analysis suggests that the high alkali concentration increases the pH of the reaction system, which is conducive to the rapid hydrolysis and uniform nucleation of metal ions, resulting in more regular nanostructures. This environment promotes the stable growth of particles, avoids excessive heterogeneity in aggregation, and ultimately improves the structural consistency and dispersibility of the products.

[0049] In summary, appropriately increasing the amount of alkali helps improve the morphology and nanostructure stability of copper ion / gallic acid nanozymes, and is one of the important means to optimize the preparation conditions.

[0050] Example 4: Effect of changing the copper salt source on morphology To investigate the effects of different copper sources on the synthesis process and structural morphology of copper ion / gallic acid nanozymes, this embodiment replaced copper nitrate trihydrate with copper chloride dihydrate while keeping the feed ratio and reaction conditions unchanged, and observed and compared the morphology of the obtained products.

[0051] The specific steps are as follows: Weigh 340.2 mg of gallic acid and 40 mg of sodium hydroxide and add them to 200 mL of deionized water. Stir thoroughly at room temperature to ensure that the solution is clear and the reactants are completely dissolved to form a complex precursor solution.

[0052] Subsequently, 341.1 mg of copper chloride dihydrate (CuCl2·2H2O) was weighed and slowly added to the above solution, and the mixture was stirred for 5 minutes to allow the copper ions to fully complex with the precursor ligand. The mixture was then transferred to a water bath at a constant temperature of 30°C and reacted for 3 hours with stirring.

[0053] After the reaction was completed, the mixture was centrifuged at 10,000 rpm for 10 minutes to separate the precipitate and discard the supernatant to obtain a copper ion / gallic acid nanozyme concentrate.

[0054] The obtained samples were observed for morphology using transmission electron microscopy (TEM). Figure 4 The image shown is a TEM image of the product of this embodiment. The results show that the product still has a spindle-shaped nanoparticle structure, but compared with the use of copper nitrate trihydrate as the copper source, the particle size distribution is slightly uneven, and slight agglomeration occurs in some areas.

[0055] Analysis suggests that the different rates at which different copper salts release copper ions in aqueous solution, the types of accompanying anions, and their coordination behavior may affect the rate and nucleation mechanism of the complexation reaction between gallic acid and copper ions, thus leading to differences in the morphology of nanozymes.

[0056] Therefore, this embodiment shows that the type of copper source has a certain regulatory effect on the structure of nanozymes, and the appropriate selection of copper salt is of great significance for obtaining nanozymes with regular morphology and stable structure.

[0057] Example 5: The effect of shortening the reaction time To investigate the effect of reaction time on the synthesis of copper ion / gallic acid nanozymes, this embodiment shortened the reaction time from 3 hours to 1 hour while keeping the types of raw materials, molar ratios, and reaction temperature constant, and examined the microstructural characteristics of the obtained products.

[0058] The specific steps are as follows: 340.2 mg of gallic acid and 40 mg of sodium hydroxide were added to 200 mL of deionized water and stirred with a magnetic stirrer at room temperature until completely dissolved to obtain a homogeneous and clear reaction precursor solution.

[0059] Subsequently, 483.2 mg of copper nitrate trihydrate was added to the above solution, and stirring was continued for 5 minutes to promote the initial complexation of copper ions with the precursor ligand. After thorough mixing, the solution was transferred to a 30°C water bath and heated for 1 hour with stirring.

[0060] Immediately after the reaction was complete, the reaction solution was centrifuged at 10,000 rpm for 10 minutes. The supernatant was discarded, and the precipitate was collected and prepared as a copper ion / gallic acid nanozyme concentrate.

[0061] The obtained product was observed by transmission electron microscopy (TEM). Figure 5 The nanozyme particles exhibit an irregular morphology, with some particles not fully nucleated, resulting in decreased particle size uniformity.

[0062] Analysis suggests that shortening the reaction time will result in insufficient complexation and nucleation growth of copper ions with gallic acid, leading to incomplete development of nanostructures, unstable particle morphology, and poor dispersibility.

[0063] In summary, this embodiment demonstrates that reaction time is one of the important parameters for controlling the morphology and dispersibility of nanozyme particles. Too short a reaction time is detrimental to the formation of structurally complete and uniformly distributed nanozyme particles, affecting their subsequent enzyme activity and application performance.

[0064] Example 6: Optimized conditions for moderate reaction time To further optimize the preparation conditions of copper ion / gallic acid nanozymes, this embodiment sets the reaction time to 2 hours based on the standard formulation to evaluate the effect of a moderate reaction time on the product structure and morphology, and to explore the optimal balance point between short time (1 hour) and long time (3 hours).

[0065] The specific steps are as follows: Weigh 340.2 mg of gallic acid and 40 mg of sodium hydroxide and add them to 200 mL of deionized water. Stir to dissolve until a clear and homogeneous reaction solution is formed.

[0066] Subsequently, 483.2 mg of copper nitrate trihydrate was added to the solution, and the mixture was stirred for 5 minutes to ensure that the copper ions and organic ligands were fully mixed and initially complexed.

[0067] The mixture was transferred to a water bath at a constant temperature of 30°C and stirred for 2 hours to promote the full reaction, nucleation and growth of copper ions and gallic acid under stable conditions.

[0068] After the reaction is complete, the reaction solution is immediately removed and centrifuged at 10,000 rpm for 10 minutes to remove the supernatant and collect the precipitate to obtain a copper ion / gallic acid nanozyme concentrate.

[0069] The prepared sample was analyzed by transmission electron microscopy (TEM), and its image is as follows. Figure 6 As shown in the figure. The results show that the nanozyme particles obtained after a reaction time of 2 hours have regular morphology, uniform particle size distribution, and uniform spindle-shaped particle structure, and have good dispersibility with no obvious agglomeration.

[0070] Compared with Example 5 (1 hour), the particles obtained in this example are more stable and have a clearer morphology; and compared with Example 1 (3 hours), their structure is also highly consistent, and the preparation cycle is shorter and more industrially efficient.

[0071] In summary, a moderate reaction time (2 hours) can appropriately shorten the reaction cycle while ensuring structural integrity and particle size uniformity, making it a preferred preparation process parameter in this invention.

[0072] Example 7: Verification of enzyme activity using the WST-8 assay To verify whether the copper ion / gallic acid nanozyme prepared in this invention possesses catalytic activity mimicking superoxide dismutase (SOD), this embodiment uses the water-soluble tetrazolium salt WST-8 colorimetric method for activity detection to evaluate its ability to scavenge superoxide anions.

[0073] The test materials and methods are as follows: The copper ion / gallic acid nanozyme sample prepared in Example 1 was selected as the test object. The SOD activity assay kit (WST-8 method) was used. This method is based on the fact that WST-8 can react with superoxide anions (O2) generated in the xanthine oxidase (XOD) system. ·-The reaction produces a soluble orange-yellow formazan dye. Its absorbance is positively correlated with the concentration of superoxide anions. When a substance with SOD-like activity is present in the sample, it can scavenge superoxide anions, inhibiting dye formation, which is manifested as a decrease in absorbance.

[0074] The experimental steps are as follows: 1. Prepare the reaction system: Add WST-8 substrate solution, XOD enzyme solution, test sample (copper / gallic acid nanozyme) sequentially to a 96-well plate, and set up a blank control group and a positive control group (natural SOD enzyme). 2. Incubate the reaction system at 37°C for 30 minutes; 3. Measure the absorbance of each well at a wavelength of 450 nm using an ELISA reader; 4. Calculate the SOD activity inhibition rate and unit activity of the sample.

[0075] The results showed that, at a sample concentration of 0.55 μg / mL, the copper ion / gallic acid nanozyme prepared in Example 1 had an average inhibition rate of 62.8% against superoxide anion, corresponding to a simulated SOD activity of 10945 Units / mg.

[0076] This result demonstrates that the copper ion / gallic acid nanozyme prepared in this invention can significantly scavenge O2 even at low concentrations. ·- It exhibits excellent superoxide dismutase activity. Compared to natural enzymes, it is more stable and easier to prepare, making it suitable for antioxidant therapy of oxidative stress-related diseases.

[0077] Figure 7 The colorimetric response of the copper / gallic acid nanozyme obtained by WST-8 assay to the superoxide anion inhibition capacity at different concentrations was demonstrated, further confirming its enzymatic properties.

[0078] Example 8: Electron paramagnetic resonance verification of free radical scavenging effect To further verify the effect of the copper ion / gallic acid nanozyme of the present invention on superoxide anion (O2) · In addition to colorimetric methods, this embodiment uses electron paramagnetic resonance (ESR) spectrometry to detect free radicals and analyze their free radical scavenging effect in a more intuitive and quantitative way.

[0079] Experimental principle: ESR (Electroradiographic Reduction) is a highly sensitive method for detecting free radical signals containing unpaired electrons. When a free radical reacts with a spin trapping agent, it forms a stable spin adduct, the characteristic spectrum of which can be detected by an ESR instrument. If a nanozyme has the ability to scavenge free radicals, the ESR signal intensity will be significantly reduced after the sample is added.

[0080] The experimental steps are as follows: 1. Preparation of free radical generation system: Riboflavin (photosensitizer) and spin trapping agent DMPO (dimethylpyrrolidone-N-oxide) are added to methanol solution to form a standard free radical reaction system; 2. Irradiation to generate free radicals: The solution was irradiated with an ultraviolet lamp with an excitation wavelength of 365 nm to induce riboflavin to generate superoxide anions; 3. Group experiments: Control group: No copper ions / gallic acid nanozymes added; Experimental group: Added copper ion / gallic acid nanozyme prepared in Example 1; 4. Measurement: Each group of solutions was injected into a quartz capillary tube, and the signal intensity was detected using an electron paramagnetic resonance spectrometer; 5. Analysis results: Compare the changes in the morphology and intensity of the free radical signal peaks.

[0081] The results show that in the control group without nanozymes, the ESR spectrum exhibited a typical six-peak superoxide anion signal with stable intensity, indicating the presence of a large number of free radicals.

[0082] In the experimental group with added copper ions / gallic acid nanozymes, the sixtet signal in the ESR spectrum was significantly weakened and the overall intensity decreased, indicating that free radicals were effectively scavenged. Figure 8 The spectrum shown intuitively illustrates the trend of signal changes.

[0083] This embodiment further verifies that the copper ion / gallic acid nanozyme prepared in this invention possesses excellent superoxide anion scavenging ability and exhibits bioactivity mimicking superoxide dismutase. Compared with traditional enzymes, this nanozyme demonstrates significant advantages in stability, tunability, and ease of synthesis, and possesses broad potential for biomedical applications.

[0084] Through the detailed descriptions of Examples 1 to 8, it can be seen that the copper ion / gallic acid nanozyme provided by the present invention has good preparation flexibility and parameter adjustability. Different alkali feed ratios (Examples 2 and 3) have a significant impact on product size and dispersibility; changes in the type of copper salt (Example 4) demonstrate the role of the metal precursor in controlling nucleation behavior and morphology; and adjustments in reaction time (Examples 5 and 6) further illustrate the contribution of the reaction process to particle structure stability.

[0085] Furthermore, the enzymatic function of this nanozyme in mimicking natural superoxide dismutase (SOD) was verified by both WST-8 colorimetry and electron paramagnetic resonance technology (Examples 7 and 8), confirming its significant activity in free radical scavenging.

[0086] The above embodiments not only cover the entire process from preparation technology to functional verification, but also fully demonstrate the diversity and controllability of the technical path of this invention. All proposed variations do not depart from the basic principles of this invention, reflecting the broad adaptability and engineering promotion value of the technical solution.

[0087] In summary, this invention provides a copper ion / gallic acid nanozyme with superoxide dismutase activity, its preparation method, and its applications. By optimizing the molar ratio of copper ions, gallic acid, and alkali, and controlling the reaction time and temperature under mild conditions, a nanozyme material with stable structure, controllable particle size, and good dispersibility can be prepared. This nanozyme not only possesses good physical stability but also exhibits excellent superoxide dismutase activity, effectively scavenging superoxide anions and alleviating oxidative stress.

[0088] Compared with the prior art, the present invention has the following significant technical effects and advantages: 1. The preparation method is simple to operate, the conditions are mild, and it is suitable for large-scale production; 2. The raw materials used are economical and readily available, and have good prospects for industrialization; 3. The obtained product has high enzyme activity and good SOD mimicry function; 4. Particle size and morphology are controllable, and the stability is strong; 5. The raw materials are low in toxicity and have good biocompatibility, making them suitable for applications in multiple fields such as medicine and pharmacy; 6. It has a wide range of applications and can be used in multiple fields such as antioxidant drugs, disease intervention agents, biological detection, and functional materials.

[0089] This invention not only expands the research path of nanozymes from the perspective of biomimetic catalysis, but also provides a new material basis for the treatment of oxidative stress-related diseases, possessing high academic research value and industrial transformation potential.

[0090] It should be understood that although this specification has described the invention in detail through multiple embodiments, these embodiments are only for illustration and to help understand the technical solutions of the invention. For those skilled in the art, various equivalent modifications or substitutions can be made without departing from the basic concept of the invention, and all such modifications or substitutions should be included within the protection scope of the invention.

Claims

1. A copper ion / gallic acid nanozyme with superoxide dismutase activity, characterized in that, The nanozyme is formed by the reaction of copper ions, gallic acid and base, wherein the molar ratio of copper ions, gallic acid and base is 1:1:0.5 to 1:1:2, and the nanozyme has the function of scavenging superoxide anions.

2. The copper ion / gallic acid nanozyme according to claim 1, characterized in that, The alkali is sodium hydroxide.

3. A method for preparing the copper ion / gallic acid nanozyme according to claim 1 or 2, characterized in that, Includes the following steps: Dissolve gallic acid and base in deionized water and stir until dissolved; Add water-soluble copper salt and continue stirring to mix; React at 30℃ for 1-3 hours; After the reaction is complete, centrifuge and collect the precipitate to obtain the copper ion / gallic acid nanozyme.

4. The preparation method according to claim 3, characterized in that, The water-soluble copper salt is copper nitrate trihydrate or copper chloride dihydrate.

5. The preparation method according to claim 3, characterized in that, The centrifugation conditions were 10,000 rpm for 10 minutes.

6. The use of the copper ion / gallic acid nanozyme according to claim 1 or 2 in the preparation of a medicament for treating oxidative stress-related diseases.

7. The application according to claim 6, characterized in that, The oxidative stress-related diseases include inflammatory diseases, cardiovascular and cerebrovascular diseases, or neurodegenerative diseases.

8. The application according to claim 7, characterized in that, The inflammatory disease is periodontitis or glomerulonephritis; the cardiovascular and cerebrovascular disease is ischemia-reperfusion injury or atherosclerosis; the neurodegenerative disease is Alzheimer's disease or Parkinson's disease.

9. The use of the copper ion / gallic acid nanozyme according to claim 1 or 2 in the preparation of a formulation for scavenging superoxide anions.

10. A pharmaceutical composition, characterized in that, It comprises the copper ion / gallic acid nanozyme as described in claim 1 or 2, and a pharmaceutically acceptable carrier.

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