Tannic acid powder containing metal-polyphenol nano enzyme as well as preparation method and application of tannic acid powder

Abstract

The invention discloses tannic acid powder containing metal-polyphenol nano-enzyme as well as a preparation method and application thereof, and relates to the technical field of medical biological materials, the powder is prepared from cerium-tannic acid nano-enzyme, tannic acid, chitosan and corn starch according to a specific ratio; the preparation method comprises the following steps: carrying out coordination self-assembly on tannic acid and cerium ions to form nano-enzyme, and mixing the nano-enzyme with other components to prepare powder. According to the powder, the peroxidase-like catalytic activity of the nano-enzyme is utilized to generate sterilization free radicals, and the efficient and drug-resistant antibacterial effect is achieved; meanwhile, the anti-inflammatory and anti-oxidation characteristics of the tannic acid and the hemostasis and healing promotion functions of the chitosan are achieved, and the multiple effects of hemostasis, antibiosis, anti-inflammation and wound healing promotion are achieved. The preparation method has the advantages of easily available raw materials, simple process, low cost, good biocompatibility and high stability, is suitable for large-scale production, and has a good application prospect in animal clinical wound treatment.

Description

Technical Field

[0001] This invention belongs to the field of medical biomaterials technology, and particularly relates to a tannic acid powder containing metal-polyphenol nanozymes, its preparation method and application. Background Technology

[0002] Tannic acid (TA) is a natural plant polyphenol widely found in trees and higher plants, extracted from gallnuts. Its molecular structure contains abundant phenolic hydroxyl groups, endowing it with various biological activities such as antioxidant, anti-inflammatory, antibacterial, and antiviral properties. In the field of biomaterials, tannic acid has attracted attention due to its good biocompatibility and diverse functional properties, showing particular potential in wound dressings and topical treatments. Furthermore, tannic acid plays an important role in stabilizing nanoparticles, improving drug bioavailability, and enhancing efficacy. However, tannic acid itself has a large molecular weight and strong hydrophilicity. When used topically, excessively high concentrations may irritate mucosal tissues, affecting its safety and tolerability in wound treatment. Simultaneously, tannic acid is easily oxidized in liquid environments and has poor stability, limiting its long-term storage and practical application effectiveness. Therefore, improving the release properties of tannic acid, reducing its local irritation, and enhancing its stability have become important issues for promoting its clinical translation.

[0003] In recent years, the self-assembly of metal ions and polyphenolic compounds based on coordination bonds to form metal-polyphenol nanocomplexes has become an effective functionalization modification strategy. These nanocomplexes not only possess good biocompatibility and tunable catalytic properties, but also achieve highly efficient antibacterial activity at low concentrations, thus avoiding the cytotoxicity problems associated with high concentrations of tannins. Of particular note is that these materials can exhibit catalytic activity similar to natural enzymes, and are termed "nanozymes." They disrupt the bacterial environment through catalytic reactions, making it less likely to induce antibiotic resistance, thus providing a new approach to addressing the current serious problem of antibiotic resistance.

[0004] Among the many metal ions, cerium ions (Ce) are particularly important. 3+ / Ce 4+ Due to its variable valence state and good biocompatibility, cerium tannin (CeTA) nanonetwork, formed through self-assembly from tannic acid and cerium nitrate, exhibits a variety of enzyme-like catalytic properties. Based on the inversion of cerium ion valence states, CeTA nanozymes show significant peroxidase (POD)-like activity, capable of catalyzing the decomposition of hydrogen peroxide (H₂O₂) to generate hydroxyl radicals (·OH). These radicals possess strong oxidizing power, efficiently disrupting the integrity of bacterial cell membranes, oxidizing intracellular proteins and nucleic acids, thereby inhibiting bacterial proliferation and even directly killing bacteria. This provides core efficacy support for the treatment of infected wounds and combating bacterial resistance.

[0005] Although CeTA nanozymes exhibit excellent antibacterial properties and catalytic activity in vitro, their practical application still faces a series of challenges. First, the stability of nanozymes in the dry state and during long-term storage needs further improvement. Second, pure nanozyme powder is not easily dispersed and adhered to the wound site, affecting its full therapeutic effect. Furthermore, an ideal wound treatment agent should possess multiple functions including hemostasis, antibacterial activity, anti-inflammation, and promoting healing; single-component nanozymes cannot meet this comprehensive requirement. Therefore, developing a topical formulation that combines the highly efficient catalytic antibacterial properties of nanozymes with other bioactive components, while possessing good stability, ease of use, and comprehensive therapeutic functions, is of significant practical importance.

[0006] In terms of topical formulations, powders offer advantages such as large coverage area, ease of storage and transportation, convenient use, and the ability to absorb wound exudate and form a protective layer, making them particularly suitable for treating skin, mucous membranes, and irregular wounds in animal clinics. Currently, some wound powders based on chitosan, gelatin, or other natural polymers have been reported, but they either focus on physical coverage and moisturizing or have limited antibacterial efficacy, especially against drug-resistant bacteria. The integration of CeTA nanozymes with highly efficient enzyme-catalyzed antibacterial properties with natural ingredients possessing anti-inflammatory and healing-promoting functions into a compound powder, and the subsequent scientific formulation optimization to construct a multifunctional synergistic treatment system, has not yet been publicly reported.

[0007] In summary, although tannic acid and metal-polyphenol nanozymes have each shown potential in the field of wound treatment, how to overcome the limitations of single components through material design and formulation process innovation, and develop a compound powder that is simple to prepare, stable in performance, and has efficient antibacterial, anti-inflammatory, hemostatic and healing-promoting functions, remains a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention proposes a tannic acid powder containing metal-polyphenol nanozymes, its preparation method, and its application. This method involves autonomously loading natural polyphenolic tannic acid and cerium ions through coordination bonds to obtain CeTA nanozymes. These are then combined with excipients such as chitosan. Through a defined powder preparation process and orthogonal experiments, a compound tannic acid powder is prepared. This powder possesses excellent properties such as simple preparation process, good formulation stability, convenient use, low cost, high biocompatibility, hemostasis, antibacterial properties, and healing promotion.

[0009] To achieve the above objectives, the present invention provides a cerium-tannic acid nanozyme, the preparation method of which includes the following steps: tannic acid and cerium nitrate hexahydrate are dissolved in deionized water at a molar ratio of 1:6 to 1:800 respectively; the cerium salt solution is added dropwise to the tannic acid solution under stirring; then the pH of the mixture is adjusted to 7.5-8.5 using an alkaline solution; the reaction is continuously stirred at room temperature for 2-4 hours; after centrifugation, washing, and vacuum freeze-drying, a dark brown cerium-tannic acid nanozyme powder is obtained.

[0010] Furthermore, the molar ratio of tannic acid to cerium nitrate hexahydrate is 1:100; the alkaline solution is a 2 M sodium hydroxide solution, adjusted to pH 8.0; the reaction time is 3 hours; the centrifugation speed is 8000 rpm; and the washing solvent is deionized water.

[0011] The present invention also provides a tannic acid compound powder containing metal-polyphenol nanozymes, which is composed of the following components by mass percentage: 10%~20% of the above-mentioned cerium-tannic acid nanozymes, 5%~20% of tannic acid, 5%~20% of chitosan, and the balance of corn starch.

[0012] Furthermore, the mass percentages of each component are as follows: cerium-tannic acid nanozyme (CeTA) 10%, tannic acid (TA) 20%, chitosan (CS) 5%, and corn starch 65%.

[0013] Furthermore, the molar ratio of tannic acid to cerium ions in the cerium-tannic acid nanozyme (CeTA) is 1:6 to 1:800, preferably 1:100.

[0014] This invention also provides a method for preparing the tannic acid compound powder containing metal-polyphenol nanozymes as described above, comprising the following steps: (1) Preparation of cerium-tannic acid nanozyme: Tannic acid and cerium salt are dissolved in deionized water and mixed to form a reaction solution. The pH of the reaction solution is adjusted to 7.5-8.5 and stirred at room temperature for 2-4 hours. After the reaction is completed, the cerium-tannic acid nanozyme powder is obtained by centrifugation, washing and freeze-drying. (2) Raw material pretreatment: The cerium-tannic acid nanoenzyme powder, tannic acid, chitosan and corn starch obtained in step (1) are crushed and sieved respectively; (3) Mixing and dispensing: Weigh each pretreated component according to the above mass percentage and mix them evenly to obtain compound powder, and then dispense it.

[0015] Further, in step (1), the pH is adjusted using sodium hydroxide solution, and the adjusted pH value is 8.0; the stirring reaction time is 3 hours; the centrifugation speed is 7000~9000 rpm; the freeze-drying conditions are: cold trap temperature -20℃ ~ -70℃, vacuum degree below 20 Pa.

[0016] Furthermore, in step (2), the sieving is done using an 80-100 mesh sieve; in step (3), the mixing time is no less than 30 minutes.

[0017] The present invention also provides the application of the above-mentioned tannic acid compound powder containing metal-polyphenol nanozymes in the preparation of a medicine for treating wounds.

[0018] Furthermore, the wound is a skin or mucous membrane injury accompanied by bleeding, infection, or inflammation; the treatment includes hemostasis, antibacterial, anti-inflammatory, and / or wound healing promotion; preferably, the drug is a topical powder preparation for clinical wound treatment in animals.

[0019] This invention achieves full-chain technology optimization through scientific methods: in the CeTA synthesis stage, the TMB colorimetric method is used to accurately screen for TA:Ce 3+ The optimal ratio of 1:100 ensures that the peroxidase-like activity reaches its peak. During the compound formulation development stage, a three-factor, three-level orthogonal experiment determined the optimal formula of 10% CeTA, 5% chitosan (CS), and 20% TA. CeTA acts as the core antibacterial agent, exerting a strong bactericidal effect. TA possesses both antioxidant and auxiliary antibacterial properties. Food-grade chitosan synergistically combats bacteria and improves formulation stability. Pharmaceutical-grade corn starch serves as a filler and dispersant, ensuring the powder's flowability and uniformity. The complementary functions of each component form a multidimensional therapeutic system of "antibacterial + anti-inflammatory + healing-promoting." The minimum inhibitory concentration (MIC) of this compound against Escherichia coli is only 12.5 mg / mL, and the minimum bactericidal concentration (MBC) is 25 mg / mL, achieving a 100% inhibition rate and demonstrating excellent antibacterial efficacy.

[0020] In terms of formulation, this invention adopts a topical powder dosage form. The preparation process follows a simple flow of "pulverization-sieving-mixing-dosing-packaging," requiring no special high-end equipment. It operates under mild conditions and is easy to scale up, reducing production costs and offering advantages such as stable storage, convenient transportation, and portability. Its large coverage area allows for simultaneous protection and astringent effects, combined with hemostasis and wound healing promotion, making it directly applicable to various scenarios such as animal skin, mucous membranes, and wound infections, meeting diverse needs in livestock farming, pet healthcare, and other fields. The core raw materials and excipients are all derived from natural plants or food / pharmaceutical grade ingredients, with wide availability, low cost, and good biocompatibility, completely avoiding the potential toxic side effects and environmental residue risks associated with chemically synthesized antibacterial agents.

[0021] Compared with the prior art, the present invention has the following advantages and technical effects: This invention uses tannic acid, an extract of the natural plant Galla chinensis, as the main raw material. It self-assembles with cerium ions via coordination bonds to prepare cerium-tannic acid nanozymes. Then, it is combined with excipients such as chitosan. Through a defined powder preparation process and orthogonal experiments, a compound tannic acid powder is prepared. The compound tannic acid powder prepared by this invention improves the stability and biocompatibility of the nanozyme, enhances the stability of natural polyphenols, and retains their antioxidant properties. Furthermore, while retaining the anti-inflammatory properties of tannic acid, this compound powder also exhibits excellent antibacterial properties. The topical powder formulation can also achieve hemostasis and promote healing. Therefore, this compound powder possesses excellent properties such as simple preparation process, good formulation stability, convenient use, low cost, high biocompatibility, hemostasis, antibacterial activity, and wound healing promotion, making it of significant application value in animal clinical wound treatment. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a schematic diagram (UV-Vis absorption spectrum) showing the peroxidase (POD) activity assay results of CeTA nanozymes prepared in different molar ratios in Example 1.

[0024] Figure 2 This is a transmission electron microscope (TEM) image of the CeTA nanozyme prepared according to the optimal molar ratio in Example 1.

[0025] Figure 3 The elemental distribution map of the CeTA nanozyme prepared in Example 1 is the energy dispersive X-ray spectroscopy (EDS) map, including the elemental distribution map of Ce, C, and O, and the superimposed map of the three.

[0026] Figure 4 This is a schematic diagram showing the POD activity results of CeTA at different pH values ​​in Comparative Example 1.

[0027] Figure 5 This is a schematic diagram showing the comparison of the antibacterial effects of a single component and a mixture in Comparative Example 2.

[0028] Figure 6 This is a schematic diagram illustrating the antibacterial effect of the optimal formulation powder in Example 4 against Escherichia coli.

[0029] Figure 7 This is a schematic diagram comparing the antibacterial effects of the composite powder in Comparative Example 3 with those of commercially available chitosan hemostatic powder.

[0030] Figure 8 This is a schematic diagram of the cytotoxicity (biocompatibility) test results of CeTA nanozyme in Example 5.

[0031] Figure 9 This is a schematic diagram of the results of the verification test on the healing effect of CeTA nanozyme compound powder in Example 6. Detailed Implementation

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] All raw materials used in this invention are not particularly restricted in their source; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0034] There are no particular restrictions on the purity of any of the raw materials used in this invention. However, this invention preferably uses raw materials of analytical grade or purity commonly used in the field of chemical synthesis.

[0035] Example 1: Preparation and Characterization of CeTA Nanozymes This embodiment aims to illustrate the optimal synthesis conditions and structural characterization of CeTA nanozymes. The specific steps are as follows: (1) At room temperature, weigh 21.6 mg of tannic acid (TA) powder, dissolve it in 30 mL of deionized water, place it in a 100 mL beaker, and stir magnetically until completely dissolved to obtain a clear TA solution. Separately, weigh 0.5513 g of cerium nitrate hexahydrate (Ce(NO3)3·6H2O), dissolve it in 10 mL of deionized water, and stir to dissolve to obtain colorless and transparent Ce. 3+ Solution. Under continuous magnetic stirring (500 rpm), Ce... 3+ The solution is added slowly, drop by drop, to the TA solution using a dropping funnel. At this point, TA and Ce... 3+ The molar ratio is 1:100.

[0036] (2) Prepare a 2 M sodium hydroxide (NaOH) solution as a pH adjuster. While continuously stirring, slowly add the NaOH solution dropwise to the mixture obtained in step (1), and monitor the pH value of the solution in real time. As NaOH is added, the mixture gradually changes from clear to a dark purple suspension. Adjust the final pH to 8.0.

[0037] (3) Continue stirring the above mixture at room temperature for 3 hours to ensure that cerium ions and tannic acid molecules fully self-assemble through coordination bonds.

[0038] (4) After the reaction is complete, transfer the suspension to a centrifuge tube and centrifuge at 8000 rpm for 10 minutes to collect the precipitate. Discard the supernatant, resuspend the precipitate in deionized water and wash by centrifugation. Repeat this process 3 times to thoroughly remove unreacted ions and byproducts. Freeze-dry the washed precipitate under vacuum (cold trap temperature -50℃, vacuum degree <10 Pa) to obtain dark brown, fluffy CeTA nanozyme powder, which is then stored in a desiccator for later use.

[0039] To determine the optimal synthesis ratio, this embodiment also sets different TA:Ce ratios. 3+ The control experiments with molar ratios of 1:6, 1:50, 1:100, 1:200, 1:400 and 1:800 were conducted, with the same synthesis steps as above.

[0040] The peroxidase-like (POD) activity of CeTA nanozymes synthesized at different ratios was evaluated using the TMB (3,3',5,5'-tetramethylbenzidine) colorimetric method. In a 3 mL acetate-sodium acetate buffer (pH 4.5) system, 100 μL of CeTA nanozyme dispersions (1 mg / mL), 500 μL of hydrogen peroxide (H₂O₂, 10 mM), and 200 μL of TMB solution (2 mM) at different ratios were added sequentially. After reacting at room temperature for 5 minutes, the absorbance (A652) of the reaction system at 652 nm was immediately measured using a UV-Vis spectrophotometer.

[0041] The results are as follows Figure 1 As shown, all CeTA synthesized in all ratios exhibited a characteristic absorption peak at 652 nm, indicating that they all possess POD activity. Specifically, when TA:Ce... 3+ When the molar ratio is 1:100, A 652nm The value reaches its highest, indicating that CeTA synthesized at this ratio exhibits the strongest POD activity. Therefore, the optimal synthesis ratio is determined to be TA:Ce. 3+ = 1:100.

[0042] The morphology of CeTA synthesized at the optimal ratio (1:100) was characterized using transmission electron microscopy (TEM). Figure 2 As shown, CeTA exhibits as uniformly sized and well-dispersed ultra-small nanoparticles with an average particle size of approximately 10 nm, and no obvious agglomeration, indicating that the synthesis method can obtain nanomaterials with good morphology.

[0043] Elemental surface scanning analysis was performed using energy-dispersive X-ray spectroscopy (EDS), and the results are as follows: Figure 3As shown, Ce, C, and O are uniformly distributed in the nanoparticles, confirming the successful complexation of Ce with TA, forming a uniform metal-polyphenol coordination network.

[0044] Comparative Example 1: Effect of changing the reaction pH on POD activity To illustrate the criticality of the reaction pH, this comparative example uses a TA:Ce reaction. 3+ CeTA was synthesized under a 1:100 ratio, and acetate-sodium acetate buffer solutions with different pH values ​​were prepared. Other steps were the same as in Example 1, using the TMB method to test POD activity. Results Figure 4 The results showed that CeTA exhibited POD activity in a slightly acidic environment, with stronger enzyme-like activity occurring in more acidic conditions. As the pH of the reaction environment increased, the ability of POD to generate free radicals gradually decreased. This indicates that the slightly acidic environment of the wound during bacterial infection promotes the production of ROS by nanozymes for bactericidal action. As the wound heals and the pH rises, the nanozymes cease ROS production, achieving bactericidal action while dynamically adapting to the wound healing microenvironment, providing a safe and efficient treatment option for infected wounds.

[0045] Example 2: Preliminary Formulation Design and Preparation Process of Compound Powder Based on literature review and single-factor preliminary experiments, this embodiment preliminarily determined the composition and basic preparation process of the compound powder. The preliminary component ratios are as follows: CeTA nanozyme 10%–20%, tannic acid (TA) 5%–20%, chitosan (CS, degree of deacetylation ≥90%, food grade) 5%–20%, and corn starch (pharmaceutical grade) to 100%. This serves as the reference starting point for subsequent orthogonal experimental design.

[0046] Its preparation process is as follows: (1) Grinding and sieving: CeTA freeze-dried powder, tannic acid powder, chitosan powder and corn starch are initially ground in a mortar (preferably a quartz mortar). Then, all raw materials are passed through an 80-mesh standard sieve to remove large particles and impurities, ensuring that the particle size of the raw materials is basically consistent, which is conducive to uniform mixing in the subsequent process.

[0047] (2) Mixing: According to the above preliminary ratio, accurately weigh each component after sieving, place them in a three-dimensional motion mixer or V-shaped mixing drum, and mix for 30-60 minutes until each component is evenly distributed in the powder and has a consistent color.

[0048] (3) Dosage and Packaging: Dispense the well-mixed powder into predetermined doses (e.g., 5g / bottle, 10g / bag) in an environment with relative humidity <40%. Use aluminum foil bags or brown glass bottles for sealed packaging to avoid moisture absorption and light exposure.

[0049] (4) Quality inspection: The finished powder should be inspected for appearance. It should be a dry, loose, free-flowing uniform powder with a uniform light brown color, free of lumps and foreign matter.

[0050] The process is simple, requires no high temperature, high pressure or complex chemical reactions, and operates under mild conditions, making it easy to scale up production.

[0051] Example 3: Screening the optimal ratio of compound powder through orthogonal experiments To obtain the optimal compound powder formulation for antibacterial performance, this embodiment uses CeTA content (A), chitosan content (B), and tannic acid content (C) as three factors for investigation, with three levels set for each factor, and designs an L9(3) formulation. 4 Orthogonal experimental design. Factor level design is shown in Table 1 below: Table 1 Nine batches of compound powder samples were prepared according to the nine proportions shown in Table 2 of the orthogonal array, with the remaining portion supplemented with corn starch to 100%, following the process described in Example 2.

[0052] Table 2 The optimal formulation was screened using the inhibition rate against *E. coli* (ATCC 25922) as the evaluation index. The test method was as follows: Each powder sample was prepared into a suspension with a concentration of 50 mg / mL using phosphate-buffered saline (PBS). The activated *E. coli* bacterial suspension was diluted to a concentration of approximately 1 × 10⁻⁶. 6 CFU / mL. Mix 1 mL of bacterial culture with 1 mL of powder suspension in a sterile centrifuge tube and incubate at 37°C with shaking for 6 hours. After incubation, take the supernatant and perform appropriate serial dilutions with PBS. Spread 100 μL of the diluted solution onto LB agar plates and incubate at 37°C for 16-18 hours before counting colonies. Use the treatment group with only PBS and no powder as the positive control (PC). Calculate the inhibition rate for each group. Inhibition rate (%) = [(Positive control group colony count - Experimental group colony count) / Positive control group colony count] × 100%.

[0053] The results of the orthogonal experiment and the range analysis are shown in Table 3 below: Table 3 Based on the results in Table 3, range (R) analysis was performed on each factor. The range R value reflects the order of influence of the factor on the antibacterial rate. The results show that RC > RA ≈ RB, meaning that the content of tannic acid (TA) has the greatest impact on the antibacterial rate of the powder, followed by the content of CeTA and chitosan, and the influence of the two is similar. By comparing the mean k at each factor level, the optimal level combination can be determined to be A1B1C3, that is, CeTA 10%, chitosan 5%, and tannic acid 20%. This combination (corresponding to group 2 and group 4 in the orthogonal experiment) achieved an antibacterial rate of 100%. Therefore, the optimal mass ratio of the compound powder was determined to be: CeTA 10%, TA 20%, CS 5%, and corn starch 65%.

[0054] Comparative Example 2: Comparison of antibacterial effects between single components and mixtures To illustrate the synergistic effect of the compound formulation, a slightly acidic physiological environment similar to that of bacterial infection was simulated. The activated *E. coli* bacterial culture was washed with PBS (pH=6) and diluted to a concentration of approximately 1×10⁻⁶. 6 CFU / mL was mixed with the powder suspension (final concentration 2 mg / mL) in a sterile centrifuge tube and incubated at 37°C with shaking for 6 hours. After incubation, the supernatant was serially diluted with PBS, and 100 μL of the diluted solution was spread onto LB agar plates and incubated at 37°C for 16-18 hours before colony counting. The treatment group with only PBS and no powder was used as the positive control (PC), and the inhibition rate of each group was calculated. The following comparative ratios were set up: Comparative Example A: Only 20% TA + 80% corn starch were used.

[0055] Comparative Example B: Using only 10% CeTA + 90% corn starch.

[0056] Comparative Example C: Using only 5% CS + 95% corn starch.

[0057] Optimal formulation: Physically mix 10% CeTA, 20% TA, 5% CS, and 65% corn starch.

[0058] The inhibition rates of single components and mixtures against *Escherichia coli* were tested according to the method in Example 3. The results are shown below. Figure 5 The antibacterial rate of comparative example A was 60%, comparative example B was 80%, and comparative example C was 99%, while the antibacterial rate of the compound powder of the optimal formulation of this invention was 100%. This indicates that the components produced a significant antibacterial synergistic effect under the specific ratio and process of this invention, and that the refined mixing process is crucial to the performance.

[0059] Example 4: Evaluation of the in vitro antibacterial properties of the optimal formulation powder The compound powder was prepared according to the optimal ratio determined in Example 3 (CeTA 10%, TA 20%, CS 5%, corn starch 65%). Its antibacterial efficacy against Escherichia coli was evaluated.

[0060] Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC): The powder was prepared into suspensions with a series of concentration gradients (25, 12.5, 6.25, 3.125 mg / mL) using PBS. 100 μL of bacterial suspension (1×10⁻⁶ mg / mL) was then used. 6 Mix (CFU / mL) with an equal volume of powder suspensions of different concentrations in a 96-well plate and incubate at 37°C for 6 hours. The lowest powder concentration at which no visible colonies grow is defined as the MIC. Then, take 10 μL of the mixture from each well and inoculate it onto fresh LB agar plates. Incubate at 37°C for 18 hours. Spread all the mixture from the wells with concentrations above the MIC onto plates. After incubation, the lowest concentration that kills more than 99.9% of bacteria (colon count ≤10) is defined as the MBC.

[0061] like Figure 6 As shown, the antibacterial rate of the powder increased significantly with increasing concentration. For Escherichia coli, the MIC was 12.5 mg / mL and the MBC was 25 mg / mL, indicating that the powder has a strong antibacterial effect.

[0062] Comparative Example 3: Comparison of antibacterial effects between compound powder and commercially available chitosan hemostatic powder A commercially available chitosan hemostatic powder (mainly composed of chitosan) was selected as a control and compared in parallel with the optimal formulation powder of this invention. In the same antibacterial experiment against *E. coli* (powder concentrations of 25 and 12.5 mg / mL), as... Figure 7 The results showed that the commercially available chitosan powder had an antibacterial rate of 73.2% at a concentration of 12.5 mg / mL, while the antibacterial rate of the powder of this invention was 100%. The antibacterial efficacy of the product of this invention is superior to that of the control product.

[0063] Example 5: Biocompatibility evaluation of CeTA nanozyme and compound powder The cytotoxicity of CeTA nanozymes and the optimal formulation of the compound powder was evaluated using the CCK-8 assay. Mouse fibroblasts (L929) were used as the cell model.

[0064] The experimental procedure is briefly described as follows: L929 cells were cultured at a density of 5 × 10⁶ cells per well. 3Cells were seeded at a density of 100 μg / mL in 96-well plates and incubated at 37°C with 5% CO2 for 24 hours to allow cell adhesion. The original culture medium was discarded, and fresh complete culture medium containing different concentrations of CeTA nanozyme (0, 50, 100, 200, 400, 600, 800 μg / mL) or compound powder extract (converted according to powder concentration) was added to each well, with 6 replicates for each concentration. Two time groups were set up: after 24 hours and 48 hours of incubation, 10 μL of CCK-8 solution was added to each well, and incubation continued for 2 hours. The absorbance (OD) of each well at 450 nm was measured using a microplate reader. 450 Relative cell viability (%) = (Experimental group OD) 450 Mean / Control Group OD 450 (Average) × 100%.

[0065] The results are as follows Figure 8 As shown, after 48 hours, the survival rate of L929 cells remained above 100% even at a concentration as high as 800 μg / mL for CeTA nanozyme, indicating that it has excellent biocompatibility and extremely low cytotoxicity.

[0066] Example 6: Verification of the healing-promoting effect of compound powder Scratch assay: Uniform scratches were made in 6-well plates filled with L929 cells using a sterile pipette tip. After washing with PBS, the experimental group was given culture medium containing the optimal formulation powder extract (0.2 mg / mL), while the control group received only ordinary culture medium. The scratch width was observed and measured under a microscope at 0, 24, and 48 hours. The scratch healing rate after 48 hours was calculated. Figure 9 The results showed that after 48 hours, the wound healing rate of the powder group was 50.6%, which was significantly higher than that of the control group (25.5%), indicating that the powder can effectively promote fibroblast migration and has the potential to promote healing.

[0067] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A cerium-tannic acid nanozyme, characterized in that, The preparation method includes the following steps: tannic acid and cerium nitrate hexahydrate are dissolved in deionized water at a molar ratio of 1:6 to 1:

800. The cerium salt solution is added dropwise to the tannic acid solution under stirring. Then, the pH of the mixture is adjusted to 7.5-8.5 using an alkaline solution. The mixture is stirred continuously at room temperature for 2-4 hours. After centrifugation, washing, and vacuum freeze-drying, dark brown cerium-tannic acid nanozyme powder is obtained.

2. The cerium-tannic acid nanozyme according to claim 1, characterized in that, The molar ratio of tannic acid to cerium nitrate hexahydrate is 1:100; the alkaline solution is a 2 M sodium hydroxide solution, adjusted to pH 8.0; the reaction time is 3 hours; the centrifugation speed is 8000 rpm; and the washing solvent is deionized water.

3. A tannic acid compound powder containing metal-polyphenol nanozymes, characterized in that, It is composed of the following components by mass percentage: 10%~20% cerium-tannic acid nanozyme as described in claim 1 or 2, 5%~20% tannic acid, 5%~20% chitosan, and the balance corn starch.

4. The tannic acid compound powder containing metal-polyphenol nanozymes according to claim 3, characterized in that, The mass percentages of each component are as follows: cerium-tannic acid nanozyme 10%, tannic acid 20%, chitosan 5%, and corn starch 65%.

5. The tannic acid compound powder containing metal-polyphenol nanozymes according to claim 3 or 4, characterized in that, The molar ratio of tannic acid to cerium ions in the cerium-tannic acid nanozyme is 1:6 to 1:800, preferably 1:

100.

6. A method for preparing a tannic acid compound powder containing metal-polyphenol nanozymes according to any one of claims 3-5, characterized in that, Includes the following steps: (1) Preparation of cerium-tannic acid nanozyme: Tannic acid and cerium salt are dissolved in deionized water and mixed to form a reaction solution. The pH of the reaction solution is adjusted to 7.5-8.5 and stirred at room temperature for 2-4 hours. After the reaction is completed, the cerium-tannic acid nanozyme powder is obtained by centrifugation, washing and freeze-drying. (2) Raw material pretreatment: The cerium-tannic acid nanoenzyme powder, tannic acid, chitosan and corn starch obtained in step (1) are crushed and sieved respectively; (3) Mixing and dispensing: Weigh each pretreated component according to the mass percentage described in claim 3 or 4 and mix them evenly to obtain a compound powder, and then dispense it.

7. The preparation method according to claim 6, characterized in that, In step (1), the pH is adjusted using sodium hydroxide solution, and the adjusted pH value is 8.0; the stirring reaction time is 3 hours; the centrifugation speed is 7000~9000 rpm; the freeze-drying conditions are: cold trap temperature -20℃ ~ -70℃, vacuum degree below 20 Pa.

8. The preparation method according to claim 6, characterized in that, In step (2), the sieving is done using an 80-100 mesh sieve; in step (3), the mixing time is no less than 30 minutes.

9. The use of the tannic acid compound powder containing metal-polyphenol nanozymes according to any one of claims 3-5 in the preparation of a medicine for treating wounds.

10. The application according to claim 9, characterized in that, The wound is a skin or mucous membrane injury accompanied by bleeding, infection, or inflammation; the treatment includes hemostasis, antibacterial, anti-inflammatory, and / or wound healing promotion; preferably, the drug is a topical powder preparation for clinical wound treatment in animals.

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