A method for plant-mediated green synthesis of silver-cerium oxide-attapulgite multifunctional composite nanoenzymes

Abstract

This invention discloses a plant-mediated green synthesis method for silver-cerium oxide-attapulgite multifunctional composite nanoenzymes. The method involves mixing cerium nitrate and silver nitrate with an attapulgite suspension, adding honeysuckle extract, adjusting the pH, heating the reaction mixture, centrifuging, washing, drying, and then calcining under a nitrogen atmosphere to obtain the silver-cerium oxide-attapulgite multifunctional composite nanoenzyme. Cerium ions form a complex with the active ingredients in the honeysuckle extract and bind to attapulgite via hydrogen bonds. Calcination yields cerium oxide nanoparticles, while silver ions are reduced by the honeysuckle extract to obtain silver nanoparticles. This invention features a green and environmentally friendly preparation process, simple operation, and easy scale-up. The resulting nanocomposite material exhibits pH-responsive antibacterial and antioxidant multienzyme activity and can be used as an anti-drug-resistant nanomaterial in biomedicine and other fields.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials, specifically relating to a method for plant-mediated green synthesis of a multifunctional composite nanoenzyme of silver-cerium oxide-attapulgite. Background Technology

[0002] Nano-cerium dioxide has reversible Ce 3+ / Ce 4+ Redox pairs, exhibiting diverse enzymatic catalytic properties, are considered promising nanozymes with potential for disease diagnosis and treatment. The surface chemistry of cerium oxide nanoparticles is crucial to their enzyme activity; abundant surface defects and a large surface area provide sufficient surface active sites, thus offering significant peroxidase-like activity. It has been reported that cerium oxide nanoparticles with low levels of oxygen vacancy structural defects exhibit poor activity due to their weak affinity for substrates and low activation capacity for generating reactive oxygen species. Generally, the abundance of oxygen vacancy structural defects shows a significant size dependence, with smaller cerium oxide nanoparticles always introducing more oxygen vacancies. However, small-sized cerium oxide nanoparticles are prone to aggregation due to their high specific surface energy.

[0003] Studies have shown that carrier-based composite nanomaterials can regulate the physicochemical properties of metal oxide nanoparticles by overcoming their aggregation. Simultaneously, the interfacial interaction between the carrier and the metal oxide can enhance the reactivity and stability of nanozymes. Attapulgite, a uniquely structured natural one-dimensional clay mineral with distinctive porous structures and surface silanol groups, can provide nucleation sites for nanoparticles and a highly concentrated substrate microenvironment for nanozymes, facilitating substrate adsorption and transport. It can serve as a carrier for regulating the activity of cerium oxide nanoparticle enzymes. Furthermore, metal doping is also an important strategy for optimizing the enzyme activity of cerium oxide nanoparticles. Nano-silver is one of the more mature antibacterial materials currently available. Silver, as a dopant, can typically modulate the surface properties and catalytic performance of metal oxides. However, due to the significant difference in redox potentials between cerium and silver ions, the preparation methods for silver-cerium oxide composites are complex, cumbersome, and inefficient, requiring further improvement and refinement.

[0004] Plant-mediated green synthesis methods have gained significant attention in recent years due to their clean and environmentally friendly nature, as well as the safety and greenness of the products. Plant extracts typically contain active biomolecules with abundant functional groups (such as hydroxyl, carboxyl, and amino groups), including flavonoids and polyphenols. These active ingredients can not only form complexes with metal ions but also reduce and stabilize nanoparticles, offering advantages in the preparation of multi-metal or metal oxide composite nanoparticles. They enable one-pot synthesis of multi-metal-based nanoparticles, greatly simplifying the synthesis process, avoiding complex post-processing operations, and significantly improving production efficiency. Honeysuckle, an evergreen climbing shrub with a wide distribution, is a commonly used traditional Chinese medicine with a long history. It contains abundant pharmacologically active ingredients such as chlorogenic acid, isochlorogenic acid, and luteolin, possessing antibacterial, anti-inflammatory, antioxidant, and heat-clearing and detoxifying effects. The phenolic acids and flavonoids in honeysuckle have abundant phenolic hydroxyl structures, capable of chelating and reducing metal ions, and can be used as natural reagents for the green synthesis of metal and metal oxide nanoparticles. Summary of the Invention

[0005] The purpose of this invention is to provide a plant-mediated green synthesis method for a multifunctional silver-cerium oxide-attapulgite composite nanozyme. Utilizing plant-mediated green synthesis technology and attapulgite as a carrier, the method achieves one-pot synthesis of the silver-cerium oxide-attapulgite multifunctional composite nanozyme by controlling the reaction conditions through carrier interfacial interactions and the synergistic regulation of the enzyme-like catalytic activity of cerium oxide nanoparticles. The preparation process is clean, environmentally friendly, simple, and easy to operate, resulting in a highly safe product with pH-responsive, highly efficient bactericidal ability and free radical scavenging activity.

[0006] I. Preparation and Characterization of Silver-Cerium Oxide-Attapulgite Multifunctional Composite Nanozymes

[0007] This invention provides a method for plant-mediated green synthesis of silver-cerium oxide-attapulgite multifunctional composite nanozymes, comprising the following steps:

[0008] Fresh honeysuckle was washed, dried in a cool place, ground into powder, and extracted with water in a 55-75℃ water bath for 1-2 hours. The extract was then filtered to obtain honeysuckle extract. Attapulgite powder was ultrasonically dispersed in deionized water to obtain an attapulgite suspension. Cerium nitrate and silver nitrate were added, and the mixture was stirred evenly. The honeysuckle extract was then added to obtain a precursor mixture. Sodium hydroxide solution was slowly added dropwise to the precursor mixture to adjust the pH. The mixture was then heated to 55-80℃ and reacted for 2-5 hours. The product was centrifuged, washed three times with deionized water, vacuum dried, and then ground through a 200-mesh sieve. The product was transferred to a tube furnace and calcined at 300-500℃ for 1-3 hours under a nitrogen atmosphere to obtain a silver-cerium oxide-attapulgite multifunctional composite nanozyme.

[0009] The solid-liquid ratio of the honeysuckle powder to water is 1:10 to 1:100 (g / mL). The solid-liquid ratio of the attapulgite suspension is 1:6 to 1:60 (g / mL). The volume ratio of the honeysuckle extract to the attapulgite suspension is 1:1 to 5:1.

[0010] The mass ratio of cerium ions in cerium nitrate to attapulgite is 1:2.5 to 1:12; the molar ratio of silver ions in silver nitrate to cerium ions in cerium nitrate is 1:1 to 1:30.

[0011] The pH adjuster is 0.5-5 mol / L sodium hydroxide, and the pH of the reaction system is 8-11. Vacuum drying is performed at room temperature for 20-25 hours.

[0012] The synthesis mechanism of this invention is as follows: using phenolic acids and flavonoids from honeysuckle extract as reducing agents and stabilizers, and attapulgite as a carrier, a silver-cerium oxide-attapulgite composite nanozyme is synthesized in a green manner. Silver has a redox potential of +0.8V, therefore silver ions can be directly reduced by phenolic acids and flavonoids. Trivalent cerium ions readily complex with phenolic acids to form cerium complexes, which can be generated after pyrolysis. Furthermore, this invention utilizes the interfacial interaction of attapulgite nanorods and the synergistic regulation of silver nanoparticles on the enzyme-like catalytic activity of cerium oxide nanoparticles, enabling the prepared ternary composite nanozyme to possess pH-responsive peroxide enzyme activity and superoxide dismutase activity.

[0013] Figure 1 The XRD pattern of the silver-cerium oxide-attapulgite multifunctional composite nanozyme synthesized in Example 1 of this invention is shown in the figure. In addition to the characteristic diffraction peaks of attapulgite (2...), the figure also shows the characteristic diffraction peaks of attapulgite (2...). θ In addition to (e.g., 8.5°, 13.9°, 19.8°, 20.7°), in 2 θ The diffraction peaks at 28.5°, 33.1°, 47.4°, and 56.3° correspond to the (111), (200), (220), and (311) crystal planes of cerium oxide, respectively. θ The diffraction peaks at 38.1°, 44.2°, 64.4° and 77.5° correspond to the (111), (200), (220) and (311) crystal planes of silver, respectively, indicating that cerium oxide and silver nanoparticles were successfully loaded on the surface of attapulgite nanorods.

[0014] Figure 2 This is a TEM image of the silver-cerium oxide-attapulgite multifunctional composite nanozyme synthesized in Example 1. The image clearly shows...

[0015] It was observed that uniformly dispersed nanoparticles were loaded on the surface of the attapulgite nanorods. High-resolution image analysis showed that the cerium oxide nanoparticles had a particle size of about 3 nm and the silver nanoparticles had a particle size of about 5 nm. The particles were uniformly distributed and well dispersed. This result further demonstrates the successful preparation of the silver-cerium oxide-attapulgite multifunctional composite nanozyme.

[0016] Figure 3 The FTIR spectrum of the silver-cerium oxide-attapulgite multifunctional composite nanozyme synthesized in Example 1 is shown at 2924.2 cm⁻¹. -1 and 2853.7cm -1 The presence of absorption peaks for methyl and hydroxyl groups indicates that plant-based active ingredients still exist on the surface of the nanoparticles after calcination, which is beneficial to the stability of the nanoparticles; 486 cm⁻¹ −1 The strong absorption band at this point corresponds to the metal-oxygen bond, indicating the formation of cerium oxide.

[0017] II. Enzyme Activity Evaluation of Silver-Cerium Oxide-Attapulgite Multifunctional Composite Nanoenzymes

[0018] Peroxidase activity was evaluated using TMB. A simple method involved incubating a sample (3 mg / mL) in acetate-sodium acetate buffer with TMB (0.5 mmol / L) and H₂O₂ (0.5 mmol / L) at room temperature (27°C), and measuring the absorbance (652 nm) of the mixture at different time points. Kinetic parameters were acquired by varying the concentration of H₂O₂. The inhibitory effect of the nanozyme on superoxide anion was evaluated using a superoxide dismutase activity assay kit following the manufacturer's instructions. Figure 4 The figure shows the peroxidase activity evaluation results of the silver-cerium oxide-attapulgite multifunctional composite nanozyme. As can be seen from the figure, the nanozyme has excellent peroxidase activity, and the activity is strongest at pH less than 4. Figure 5 This demonstrates that the nanozyme can generate abundant hydroxyl radicals. Figure 6 The evaluation results of the superoxide dismutase activity of the silver-cerium oxide-attapulgite multifunctional composite nanozyme show that the nanozyme exhibits pH-dependent superoxide dismutase activity, with the best enzyme activity at pH greater than 6.

[0019] III. Antibacterial Activity of Silver-Cerium Oxide-Attapulgite Multifunctional Composite Nanoenzymes

[0020] The antibacterial properties of the silver-cerium oxide-attapulgite multifunctional composite nanozyme prepared in the examples were demonstrated against methicillin-resistant Staphylococcus aureus (MRSA) and extended-spectrum β-lactamase-producing Escherichia coli (ESBLs). E. coli The test strain was evaluated using the plate count method. The test method was as follows: 1) Using ESBLs revived on a petri dish... E. coliA bacterial suspension of MRSA was prepared with PBS at a concentration of 0.5 McF, resulting in a bacterial suspension concentration of approximately 1.5 × 10⁻⁶. 8 CFU / mL; dilute the bacterial suspension 100-fold to achieve a final concentration of approximately 1×10⁻⁶ CFU / mL. 6 CFU / mL; 2) Add 20µL of the sample dispersion and 50mL of buffer (pH 3.7) to each well of a 96-well plate, add 10µL of H2O2 (final H2O2 concentration is 100µM), and add 20µL of bacterial suspension; gently mix and incubate at 37°C for 2h; after incubation, take 20µL of the co-culture solution and spread it onto nutrient agar medium, incubate at 37°C for 18-24h, and count the colonies. 3) After incubation, take 20µL of the co-culture solution and spread it onto nutrient agar medium, incubate at 37°C for 18-24h, and count the colonies. The antibacterial performance evaluation results of the silver-cerium oxide-attapulgite multifunctional composite nanozymes in Examples 1-5 are shown in Table 1. It can be seen that the composite nanozymes prepared in this invention are effective against Gram-positive MRSA and Gram-negative ESBLs. E. coli The inhibition rates of both drug-resistant bacteria were above 90%, indicating that the silver-cerium oxide-attapulgite multifunctional composite nanozyme prepared in this invention effectively inhibits ESBLs. E. coli Both MRSA and other antibacterial agents exhibit highly efficient antibacterial activity.

[0021]

[0022] II. Antioxidant Properties of Silver-Cerium Oxide-Attapulgite Multifunctional Composite Nanoenzymes

[0023] The antioxidant properties of the silver-cerium oxide-attapulgite multifunctional composite nanozyme prepared in this example were evaluated using the salicylic acid method. Specifically, 1 mL of 6 mmol / L ferric sulfate heptahydrate, 1 mL of 9 mmol / L salicylic acid ethanol solution, and 1 mL of samples of different concentrations (0.1–5 mg / mL) were added to a 10 mL test tube. Then, 0.1 mL of hydrogen peroxide (0.3%) and 1 mL of ultrapure water were added to initiate the reaction. The test tube was then placed in a 37°C water bath for 30 min, and the absorbance was measured at 510 nm. Ultrapure water was used as a blank group instead of the sample, and 0.1 mL of ultrapure water was used as a control group instead of H2O2. Each measurement was repeated three times. Figure 7 This indicates that the silver-cerium oxide-attapulgite multifunctional composite nanozyme prepared in this invention has excellent hydroxyl radical scavenging activity.

[0024] In summary, the plant-mediated green synthesis of silver-cerium oxide-attapulgite multifunctional composite nanoenzymes of this invention has the following advantages:

[0025] 1. Using natural plant extracts as reducing agents and stabilizers, silver and cerium oxide composite nanoparticles can be prepared in one pot. This method is clean, environmentally friendly, simple, and produces safe and stable products. It is a clean and green preparation method.

[0026] 2. This invention effectively enhances the enzyme-like catalytic performance of cerium oxide nanoparticles by synergistic regulation of the interfacial interaction of attapulgite nanorod crystals and silver nanoparticles, thereby producing nanozymes with dual antibacterial and antioxidant effects that respond to pH. Attached Figure Description

[0027] Figure 1 The XRD pattern of the silver-cerium oxide-attapulgite multifunctional composite nanozyme prepared in Example 3 of this invention;

[0028] Figure 2 This is a TEM image of the silver-cerium oxide-attapulgite multifunctional composite nanozyme prepared in Example 3 of the present invention;

[0029] Figure 3 The FTIR image of the silver-cerium oxide-attapulgite multifunctional composite nanozyme prepared in Example 3 of this invention;

[0030] Figure 4 The peroxidase activity evaluation results of the silver-cerium oxide-attapulgite multifunctional composite nanozyme prepared in Example 3 of this invention;

[0031] Figure 5 The detection results of hydroxyl radicals generated by the silver-cerium oxide-attapulgite multifunctional composite nanozyme prepared in Example 3 of this invention;

[0032] Figure 6 The superoxide dismutase activity evaluation results of the silver-cerium oxide-attapulgite multifunctional composite nanozyme prepared in Example 3 of this invention;

[0033] Figure 7 The results of the evaluation of the hydroxyl radical scavenging activity of the silver-cerium oxide-attapulgite multifunctional composite nanoenzyme prepared in Example 3 of this invention are as follows; Detailed Implementation

[0034] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0035] Example 1

[0036] Fresh honeysuckle was washed, dried in a cool place, and ground into powder. 7g of the powder was added to 100mL of deionized water and extracted in a water bath at 60℃ for 1.5h. The extract was then filtered to obtain honeysuckle extract. 5g of attapulgite powder was ultrasonically dispersed in 30mL of deionized water to obtain an attapulgite suspension. 1.26g of cerium nitrate and 0.4g of silver nitrate were added, and the mixture was stirred evenly. Then, 90mL of honeysuckle extract was added to obtain a precursor mixture. 1M sodium hydroxide was slowly added dropwise to the above precursor mixture to adjust the pH to 9. The mixture was then heated to 80℃ and reacted for 2h. The product was centrifuged, washed three times with deionized water, and vacuum dried at room temperature for 24h. The product was then ground and passed through a 200-mesh sieve. The product was transferred to a tube furnace and calcined at 500℃ for 2h under a nitrogen atmosphere to obtain a silver-cerium oxide-attapulgite multifunctional composite nanozyme.

[0037] Example 2:

[0038] Fresh honeysuckle was washed, dried in a cool place, and ground into powder. 5g of the powder was added to 100mL of deionized water and extracted in a 55°C water bath for 2 hours. The extract was then filtered to obtain the honeysuckle extract. 0.5g of attapulgite powder was ultrasonically dispersed in 30mL of deionized water to obtain an attapulgite suspension. 0.63g of cerium nitrate and 0.008g of silver nitrate were added, and the mixture was stirred evenly. Then, 60mL of honeysuckle extract was added to obtain a precursor mixture. 0.5M sodium hydroxide was slowly added dropwise to the above precursor mixture to adjust the pH to 8. The mixture was then heated to 55°C and reacted for 5 hours. The product was centrifuged, washed three times with deionized water, and vacuum dried at room temperature for 24 hours. The product was then ground and passed through a 200-mesh sieve. The product was transferred to a tube furnace and calcined at 500°C for 2 hours under a nitrogen atmosphere to obtain a silver-cerium oxide-attapulgite multifunctional composite nanozyme.

[0039] Example 3:

[0040] Fresh honeysuckle was washed, dried in a cool place, and ground into powder. 3g of the powder was added to 100mL of deionized water and extracted in a 60°C water bath for 1.5h. The extract was then filtered to obtain honeysuckle extract. 2g of attapulgite powder was ultrasonically dispersed in 30mL of deionized water to obtain an attapulgite suspension. 1g of cerium nitrate and 0.095g of silver nitrate were added, and the mixture was stirred evenly. Then, 30mL of honeysuckle extract was added to obtain a precursor mixture. 2M sodium hydroxide was slowly added dropwise to the above precursor mixture to adjust the pH to 9. The mixture was then heated to 60°C and reacted for 3h. The product was centrifuged, washed three times with deionized water, and vacuum dried at room temperature for 24h. The product was then ground and passed through a 200-mesh sieve. The product was transferred to a tube furnace and calcined at 500°C for 2h under a nitrogen atmosphere to obtain a silver-cerium oxide-attapulgite multifunctional composite nanozyme.

[0041] Example 4:

[0042] Wash fresh honeysuckle thoroughly, dry it in a cool, shaded place, grind it into powder, weigh 9g and add it to 100mL of deionized water. Extract in a 70°C water bath for 1 hour, and filter to obtain honeysuckle extract. Disperse 3g of attapulgite powder ultrasonically into 30mL of deionized water to obtain attapulgite suspension, and add 3g...

[0043] Cerium nitrate and 0.19 g of silver nitrate were mixed evenly and then added to 120 mL of honeysuckle extract to obtain a precursor mixture. 3 M sodium hydroxide was slowly added dropwise to the above precursor mixture to adjust the pH to 10, and the mixture was heated to 70°C for 2 h. The obtained product was centrifuged, washed three times with deionized water, vacuum dried at room temperature for 24 h, and then ground through a 200-mesh sieve. The product was transferred to a tube furnace and calcined at 400°C for 2 h under a nitrogen atmosphere to obtain a silver-cerium oxide-attapulgite multifunctional composite nanozyme.

[0044] Example 5:

[0045] Fresh honeysuckle was washed, dried in a cool, and ground into powder. 10g of the powder was added to 100mL of deionized water and extracted in a 65°C water bath for 1.5 hours. The extract was then filtered to obtain the honeysuckle extract. 4g of attapulgite powder was ultrasonically dispersed in 30mL of deionized water to obtain an attapulgite suspension. 3g of cerium nitrate and 0.13g of silver nitrate were added, and the mixture was stirred until homogeneous. Then, 150mL of the honeysuckle extract was added to obtain a precursor mixture. The above precursor mixture was then added...

[0046] After slowly adding 5M sodium hydroxide to the liquid to adjust the pH to 11, the mixture was heated to 60°C and reacted for 3 hours. The resulting product was centrifuged, washed three times with deionized water, dried under vacuum at room temperature for 24 hours, and then ground through a 200-mesh sieve. The product was then transferred to a tube furnace and calcined at 300°C for 2 hours under a nitrogen atmosphere to obtain a silver-cerium oxide-attapulgite multifunctional composite nanozyme.

Claims

1. A method for plant-mediated green synthesis of silver-cerium oxide-attapulgite multifunctional composite nanozymes, comprising the following steps: Fresh honeysuckle was washed, dried in a cool place, ground into powder, and extracted with water in a 55-75℃ water bath for 1-2 hours. The extract was then filtered to obtain honeysuckle extract. Attapulgite powder was ultrasonically dispersed in deionized water to obtain an attapulgite suspension. Cerium nitrate and silver nitrate were added, and the mixture was stirred evenly. The honeysuckle extract was then added to obtain a precursor mixture. Sodium hydroxide solution was slowly added dropwise to the precursor mixture to adjust the pH. The mixture was then heated to 55-80℃ and reacted for 2-5 hours. The product was centrifuged, washed three times with deionized water, vacuum dried, ground, and sieved. The product was then transferred to a tube furnace and calcined at 300-500℃ for 1-3 hours under a nitrogen atmosphere to obtain a silver-cerium oxide-attapulgite multifunctional composite nanozyme.

2. The method for plant-mediated green synthesis of silver-cerium oxide-attapulgite multifunctional composite nanoenzymes according to claim 1, characterized in that, The solid-liquid ratio of the powder made from fresh honeysuckle to water is 1:10 to 1:

100.

3. The method for plant-mediated green synthesis of silver-cerium oxide-attapulgite multifunctional composite nanoenzymes according to claim 1, characterized in that, The solid-liquid ratio of the attapulgite suspension is 1:6 to 1:

60.

4. The method for plant-mediated green synthesis of silver-cerium oxide-attapulgite multifunctional composite nanoenzymes according to claim 1, characterized in that, The mass ratio of cerium ions in cerium nitrate to attapulgite is 1:2.5 to 1:12; the molar ratio of silver ions in silver nitrate to cerium ions in cerium nitrate is 1:1 to 1:

30.

5. The method for plant-mediated green synthesis of silver-cerium oxide-attapulgite multifunctional composite nanoenzymes according to claim 1, characterized in that, The volume ratio of the honeysuckle extract to the attapulgite suspension is 1:1 to 5:

1.

6. The method for plant-mediated green synthesis of silver-cerium oxide-attapulgite multifunctional composite nanoenzymes according to claim 1, characterized in that, The concentration of the sodium hydroxide solution is 0.5~5 mol / L, and the pH of the reaction system is adjusted to 8~11.

7. The method for plant-mediated green synthesis of silver-cerium oxide-attapulgite multifunctional composite nanoenzymes according to claim 1, characterized in that, Vacuum drying involves drying at room temperature for 20-25 hours.

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