A medical stone nano-enzyme, a preparation method and application thereof

Abstract

This invention discloses a maifanite nanozyme, prepared by mechanically activating and modifying maifanite. The maifanite nanozyme possesses the mineral components, nanoscale size characteristics, and peroxidase-like activity of maifanite. The corresponding preparation method and applications are also disclosed. This maifanite nanozyme not only serves as a trace element carrier but also endows it with previously unrecognized redox regulatory functions. Its inherent peroxidase-like activity can generate redox-mediated signal cascade reactions, acting as a redox regulator in plants, promoting root development and improving nutrient absorption, thus organically combining catalytic regulation with nutrient utilization. The maifanite nanozyme can induce systemic changes in the metabolic spectrum of plant leaves, involving multiple aspects such as amino acid metabolism, lipid metabolism, antioxidant-related metabolism, and signal molecule synthesis, providing a metabolic basis for its application in plant growth, nutrient accumulation, and quality improvement.

Description

Technical Field

[0001] This invention relates to the field of agricultural nanobiotechnology, specifically to a maifanite nanozyme, its preparation method, and its application. Background Technology

[0002] In the context of increasingly constrained environmental conditions, meeting the world's growing food demand requires sustainable strategies that go beyond traditional fertilization. The use of traditional fertilizers is limited by diminishing returns to yield and escalating ecological costs. Long-term over-fertilization not only leads to soil degradation and water eutrophication, but also, with the emergence of marginal effects, simply increasing nutrient supply is no longer sufficient to significantly improve crop yields. Therefore, current agricultural technology urgently needs a catalytic material that can precisely regulate plant metabolism, rather than simply supplying nutrients.

[0003] Nanozymes are a class of nanomaterials with enzyme-like catalytic activity. Their high stability, low cost, and ease of large-scale preparation have attracted widespread attention in the biomedical field. However, in agriculture, the development of low-cost, environmentally friendly nanozyme-based fertilizers that can effectively regulate plant physiological and biochemical processes is still in its early stages.

[0004] Maifan stone is a naturally abundant and non-toxic silicate mineral containing various trace elements required by plants. If its potential catalytic activity can be explored through specific nanotechnology techniques and it can be transformed into a functional material capable of regulating plant redox balance, it will provide a scalable pathway for next-generation sustainable agriculture. Currently, there are no reports on maifan stone nanozymes as plant redox homeostasis regulators or their effects on alleviating salt-alkali stress and increasing crop yield. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to propose a maifanite nanozyme, while providing its corresponding preparation method and application is another objective of the present invention.

[0006] A type of maifanite nanozyme, which is prepared by modifying maifanite through mechanical activation, wherein the maifanite nanozyme has the mineral composition, nanoscale size characteristics and peroxidase-like activity of maifanite.

[0007] The maifanite nanozyme has a nanoscale particle morphology with a particle size of 120-500 nm.

[0008] A method for preparing maifanite nanozymes includes the following steps: (1) Maifan stone pretreatment; (2) Homogenization: The maifan stone pretreated in step (1) is physically crushed and ground to obtain micron-sized maifan stone powder; (3) Nano-sizing treatment: The micron-sized maifanite powder from step (2) is nano-sized to obtain maifanite nanoenzymes.

[0009] In step (3), the nano-sizing method adopts high-energy ball milling, ultrasonic exfoliation or liquid phase shearing exfoliation.

[0010] In step (2), physical crushing and grinding are carried out using an impact ultrafine pulverizer. During the pulverization process, the working speed of the impact ultrafine pulverizer is 2000-3000 rpm, and it runs continuously for 3-6 times with a total running time of 20-30 min, so as to obtain maifan stone powder with a particle size of micron. In step (3), the nano-processing method employs high-energy ball milling, specifically: the micron-sized maifanite powder from step (2) is placed in a high-energy nano-impact mill, with a mass ratio of milling media to maifanite powder of approximately (2-6):1, and a filling rate of the milling media (the percentage of the milling media filling the internal volume of the milling jar) of 25-50%. The milling is performed for 40-60 minutes at a vibration frequency of 15-50 Hz and a temperature of 15-20℃, followed by sieving (filtering with steel balls) to obtain maifanite nanoenzymes. The high-energy nano-impact mill is equipped with a cooling system, using ethylene glycol as the cooling medium to maintain the temperature at 15-20℃ during the milling process.

[0011] In step (1), the pretreatment method for maifan stone is to wash the maifan stone with water 1-3 times to remove surface impurities and then dry it. In step (3), the ball milling media is composed of steel balls of different particle sizes, wherein steel balls with a diameter of 1 cm account for 50-60% of the total steel ball mass, steel balls with a diameter of 0.6 cm account for 25-30% of the total steel ball mass, and steel balls with a diameter of 0.4 cm account for 15-20% of the total steel ball mass.

[0012] Application of maifanite nanozymes in the preparation of agricultural fertilizers, plant growth regulators, or soil conditioners.

[0013] The maifanite nanoenzyme, as a component of plant growth regulators, regulates the redox homeostasis within plant cells, causing changes in the metabolic spectrum of plant leaves and promoting root development.

[0014] The plant in question is either wheat or tobacco.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1) This invention produces maifanite nanozymes by homogenizing and nano-processing natural silicate minerals. These nanozymes not only serve as trace element carriers but also endow them with previously unrecognized redox regulatory functions. Their inherent peroxidase-like activity can generate redox-mediated signal cascade reactions, acting as redox regulators in plants, promoting root development and improving nutrient absorption, thus organically combining catalytic regulation with nutrient utilization. Maifanite nanozymes exhibit catalytic stability within the physiological pH range and can induce mild oxidative stimulation in plants by regulating intracellular •OH levels, activating the plant's own antioxidant defense system and significantly enhancing the plant's adaptability to environmental stresses such as salinity and alkalinity. Maifanite nanozymes can induce systemic changes in the metabolic spectrum of plant leaves, involving multiple aspects such as amino acid metabolism, lipid metabolism, antioxidant-related metabolism, and signal molecule synthesis, providing a metabolic basis for their application in plant growth, material accumulation, and quality improvement.

[0016] 2) The maifanite nanoenzyme of the present invention can increase crop yield by about 18% under different environmental conditions, and improve crop quality by increasing nutrient accumulation.

[0017] 3) The raw material of the present invention, maifanite, is a natural, abundant, and environmentally friendly mineral. The preparation method is simple and easy to implement, providing a mechanical framework and practical path for developing low-cost and environmentally friendly nano-enzyme-based fertilizers, which helps to promote the development of sustainable agriculture. Attached Figure Description

[0018] Figure 1 The following is a description of the preparation process and physicochemical characterization results of the maifanite nanozyme in Example 1 of this invention, wherein: (a) is a schematic diagram of the process flow for preparing maifanite nanozyme by combining homogenization and nano-processing, showing the process of transforming blocky maifanite into nanoscale mineral assemblies; (b) is a scanning electron microscope (SEM) image of the prepared maifanite nanozyme; (c) is a transmission electron microscope (TEM) image of the prepared maifanite nanozyme; (d) is an energy dispersive X-ray spectroscopy (EDS) elemental distribution map of the prepared maifanite nanozyme; (e) is the analysis results of the content of each element in the prepared maifanite nanozyme; (f) is the X-ray diffraction (XRD) pattern of the prepared maifanite nanozyme; (g) is the particle size distribution and zeta potential results of the maifanite nanozyme obtained by dynamic light scattering (DLS) test; and (h) is the Fourier transform infrared (FTIR) spectrum of the prepared maifanite nanozyme.

[0019] Figure 2This is a schematic diagram of the peroxidase-like activity evaluation results of the maifanite nanozyme in Example 1 of the present invention. (a) is a schematic diagram of the peroxidase-like (POD-like) catalytic reaction mediated by the maifanite nanozyme, showing the process of the maifanite nanozyme catalyzing the decomposition of hydrogen peroxide and generating hydroxyl radicals; (b) are the UV-Vis absorption spectra of different reaction systems; (c) are the UV absorbance test results of the maifanite nanozyme at 652 nm wavelength under different pH conditions; (d) are the UV absorbance values ​​of the maifanite nanozyme at 652 nm wavelength within a temperature range of -20℃ to 50℃. (e) shows the kinetic test results of the peroxidase activity of maifanite nanoenzymes with hydrogen peroxide as the substrate; (f) shows the kinetic test results of the peroxidase activity of maifanite nanoenzymes with 3,3′,5,5′-tetramethylbenzidine (TMB) as the substrate; (g) shows the electron spin resonance (ESR) spectrum of hydroxyl radicals in the maifanite nanoenzyme system; and (h) shows the simulated activity evaluation results of maifanite nanoenzymes and the change in absorbance at 652 nm before and after catalyzing the oxidation of TMB.

[0020] Figure 3 Example 1 of the present invention is a maifanite nanoenzyme for tobacco ( Nicotiana benthamiana ) Schematic diagram of the experimental results of growth-promoting effect, wherein (a) is a schematic diagram of the hyperspectral imaging results of maifanite nanozyme in tobacco leaf cells, showing the spectral library of maifanite nanozyme, the hyperspectral image of tobacco leaf cells, and the superimposed image of the two in sequence; (b) is the detection results of silicon and aluminum content in tobacco leaves treated with and untreated with maifanite nanozyme; (c) is the detection results of silicon and aluminum content in tobacco stems treated with and untreated with maifanite nanozyme; (d) is the detection results of silicon and aluminum content in tobacco stems treated with and untreated with maifanite nanozyme. (e) Detection results of silicon and aluminum content in the roots of treated and untreated tobacco plants; (f) Statistical results of leaf number of tobacco plants; (g) Statistical results of branch number of tobacco plants; (h) Measurement results of plant height of tobacco plants; (i) Measurement results of leaf width of tobacco plants; (j) Measurement results of stem diameter of tobacco plants; (k) Detection results of chlorophyll content in tobacco plants treated and untreated with maifanite nanoenzymes.

[0021] Figure 4This is a schematic diagram illustrating the experimental results of redox regulation and transcriptional response in tobacco cells induced by maifanite nanozymes in Example 1 of the present invention. (a) is a confocal fluorescence microscopy image of the reactive oxygen species level in tobacco cells after treatment with maifanite nanozymes, with a scale bar of 20. (a) μm; (b) Superoxide dismutase (SOD) activity detection results in tobacco leaf tissue; (c) Catalase (CAT) activity detection results in tobacco leaf tissue; (d) Malondialdehyde (MDA) content detection results in tobacco leaf tissue; (e) Gene set enrichment analysis (GSEA) results of transcriptome data from tobacco leaves treated with maifanite nanoenzymes and control group; (f) Enrichment results of gene pathways related to antioxidant activity; (g) Enrichment results of gene pathways related to redox processes; (hi) Enrichment results of gene pathways related to peroxidase; (j) Enrichment results of gene pathways related to heme binding; (k) Enrichment results of metabolic pathways related to carbohydrate catabolism; (l) Enrichment results of metabolic pathways related to sulfur-containing amino acid metabolism; (m) Enrichment results of metabolic pathways related to isoprene biosynthesis.

[0022] Figure 5 This is a schematic diagram of the metabolomics analysis results of the redox-related metabolic reprogramming of tobacco leaves induced by maifanite nanozymes in Example 1 of the present invention. (a) is the principal component analysis (PCA) score plot of the non-targeted metabolomics data of tobacco leaves in the control group and the maifanite nanozyme treatment group; (b) is the volcano plot of differential metabolites between the maifanite nanozyme treatment group and the control group; (c) is the variable importance (VIP) analysis results of differential metabolites, showing the categories of metabolites that contribute significantly to the group differentiation; (d) is the KEGG metabolic pathway enrichment analysis results of differential metabolites; and (e) is the radar plot of the changes in representative differential metabolites in the maifanite nanozyme treatment group relative to the control group.

[0023] Figure 6 This is a schematic diagram of the experimental results of the effect of maifanite nanoenzyme treatment on the yield and quality of wheat throughout the entire growth period in Example 1 of the present invention. (a) is a comparison of the phenotypes of wheat plants treated with maifanite nanoenzyme foliar spray and untreated plants; (b) is the statistical result of wheat grain yield; (c) is the detection result of total protein content in wheat grain; (d) is the detection result of the content of 9 essential amino acids in wheat grain; (e) is the detection result of the content of 11 non-essential amino acids in wheat grain. Detailed Implementation

[0024] The present invention will be further described below with reference to specific embodiments and accompanying drawings. Example

[0025] A type of maifanite nanozyme is prepared by modifying maifanite through mechanical activation. The maifanite nanozyme has the mineral components, nanoscale size characteristics and peroxidase-like activity of maifanite. The maifanite nanozyme has a nanoscale particle morphology with a particle size of 120-500 nm.

[0026] A method for preparing maifanite nanozymes includes the following steps: (1) Pretreatment of Maifan stone: Wash the Maifan stone with water three times to remove surface impurities and allow it to air dry naturally; (2) Homogenization: The maifan stone pretreated in step (1) is placed in an impact ultrafine pulverizer and run continuously for 5 times at a speed of 2000 rpm for a total running time of 30 min to obtain maifan stone powder with a particle size of micron. (3) Nanoscale processing: The micron-sized maifanite powder from step (2) is placed in a high-energy nano-impact mill. The mass ratio of the milling media to the maifanite powder is approximately 3:1, and the filling rate of the milling media (the percentage of the milling media filling the internal volume of the milling tank) is 50%. The milling is carried out for 45 minutes at a vibration frequency of 40 Hz and a temperature of 15℃. After milling, the maifanite nanoenzyme is obtained by sieving through a 32-mesh filter (filtering steel balls). The high-energy nano-impact mill is equipped with a cooling system, using ethylene glycol as the cooling medium to maintain the temperature at 15℃ during the milling process. The process flow diagram is as follows. Figure 1 As shown in (a).

[0027] In step (3), the ball milling media is composed of steel balls of different sizes, wherein steel balls with a diameter of 1 cm account for 50% of the total steel ball mass, steel balls with a diameter of 0.6 cm account for 30% of the total steel ball mass, and steel balls with a diameter of 0.4 cm account for 20% of the total steel ball mass.

[0028] The physicochemical properties of the maifanite nanozyme prepared in this embodiment were characterized, and the results are as follows: Figure 1 As shown in (b)-(h). By Figure 1 (b) Scanning electron microscope and Figure 1 (c) Transmission electron microscopy observation results show that the obtained material exhibits a nanoscale particle morphology; Figure 1 (d) Energy-dispersive X-ray spectroscopy elemental analysis and Figure 1 (e) Analysis of the elemental content in the maifanite nanozyme shows that the maifanite nanozyme retains the original mineral element composition of maifanite. Figure 1 (g) Dynamic light scattering test results show that the nanozyme has nanoscale particle size distribution characteristics; Figure 1 (f) X-ray diffraction analysis shows that the material retains the crystal structure characteristics of natural minerals; Figure 1 (h) Fourier transform infrared spectroscopy results show that the material contains typical silicate structure-related absorption peaks.

[0029] The maifanite nanozymes obtained by the above preparation methods can form a stable dispersion in an aqueous system and have good structural stability, which lays the foundation for their subsequent application as a functional material with peroxidase-like activity in plant systems. Example

[0030] A type of maifanite nanozyme is prepared by modifying maifanite through mechanical activation. The maifanite nanozyme has the mineral components, nanoscale size characteristics and peroxidase-like activity of maifanite. The maifanite nanozyme has a nanoscale particle morphology with a particle size of 120-500 nm.

[0031] A method for preparing maifanite nanozymes includes the following steps: (1) Pretreatment of Maifan stone: Wash the Maifan stone with water three times to remove surface impurities and allow it to air dry naturally; (2) Homogenization: The pretreated maifan stone in step (1) is placed in an impact ultrafine pulverizer and run continuously for 3 times at a speed of 3000 rpm for a total running time of 24 min to obtain maifan stone powder with a particle size of micron. (3) Nanoscale processing: The micron-sized maifanite powder from step (2) is placed in a high-energy nano-impact mill. The mass ratio of the milling media to the maifanite powder is approximately 6:1, and the filling rate of the milling media (the percentage of the milling media filling the internal volume of the milling tank) is 25%. The milling is carried out for 60 minutes at a vibration frequency of 15 Hz and a temperature of 20℃. The maifanite nano-enzyme is obtained by sieving through a 16-mesh filter (filtering steel balls). The high-energy nano-impact mill is equipped with a cooling system, using ethylene glycol as the cooling medium to maintain the temperature at 20℃ during the milling process. The process flow diagram is as follows. Figure 1 As shown in (a).

[0032] In step (3), the ball milling media is composed of steel balls of different sizes, wherein steel balls with a diameter of 1 cm account for 60% of the total steel ball mass, steel balls with a diameter of 0.6 cm account for 25% of the total steel ball mass, and steel balls with a diameter of 0.4 cm account for 15% of the total steel ball mass.

[0033] To illustrate the peroxidase-like catalytic activity of the maifanite nanozyme in Example 1 of this invention.

[0034] The maifanite nanozyme prepared in Example 1 was dispersed in deionized water to obtain a 60 mg / mL maifanite nanozyme dispersion. A reaction system (maifanite nanozyme + TMB + hydrogen peroxide system) was constructed using H2O2 as the oxidant and 3,3′,5,5′-tetramethylbenzidine (TMB) as the chromogenic substrate, as shown in Table 1. Simultaneously, maifanite nanozyme + TMB, TMB + hydrogen peroxide, and maifanite nanozyme + hydrogen peroxide systems were also constructed. In these three systems, the concentrations and volumes of the maifanite nanozyme dispersion, TMB, and hydrogen peroxide were the same as in the maifanite nanozyme + TMB + hydrogen peroxide system in Table 1. If the total volume of the reaction system was less than 100 μL, it was made up to 100 μL with 0.2 M NaAc-Hac buffer solution. The final concentration of hydrogen peroxide (H2O2) in all three reaction systems was 500 mM.

[0035] Table 1. Reagents and dosages for the reaction system Components volume Maifan stone nanoenzyme dispersion (60 mg / mL) 10 μL <![CDATA[10 M hydrogen peroxide (H2O2)]]> 5 μL 20 mg / ml TMB (dissolved in DMSO) 1 μL 0.2 M NaAc-Hac buffer solution (pH 3.6) 84 μL Total volume 100 μL The UV-Vis absorption change of the reaction system at 652 nm wavelength was monitored within a 5-minute reaction time to characterize the peroxidase-like catalytic activity of the material. The test results are as follows: Figure 2 As shown in (b), when maifanite nanozyme, TMB, and H2O2 are present simultaneously in the reaction system, a significant change in the absorption peak at 652 nm can be detected; however, when any component is missing from the reaction system, no significant absorption peak signal is observed. These results indicate that, under specific reaction conditions, maifanite nanozyme can participate in and promote the oxidation of TMB. Furthermore, even without the addition of H2O2, some chromogenic substrates in the reaction system still undergo color changes, suggesting that maifanite nanozyme exhibits catalytic behavior similar to oxidases under certain conditions.

[0036] The effect of pH conditions on the catalytic activity of maifanite nanozymes was further investigated, and the results are as follows: Figure 2 As shown in (c), the maifanite nanozyme exhibited varying degrees of catalytic response under different pH conditions. The absorption signal was most pronounced at approximately pH 4.5, indicating higher catalytic activity under these conditions. The effect of temperature on the catalytic behavior was also investigated, and the results are shown in (c). Figure 2 As shown in (d), the catalytic reaction of the maifanite nanozyme can be maintained within a temperature range of -20℃ to 50℃, with a stronger reaction signal at approximately 40℃. Within the range of 10-30℃, the absorption changes of the reaction system are relatively stable, indicating that the maifanite nanozyme maintains relatively stable catalytic performance over a wide temperature range. Based on the enzyme-catalyzed reaction kinetic model, the peroxidase-like activity of the maifanite nanozyme was quantitatively analyzed. The reaction kinetic curve with H2O2 as a variable substrate is shown in Figure [Figure number missing]. Figure 2As shown in (e), the corresponding maximum reaction rate ( V max The value is 68.01 nM / s, and the Michaelis constant is ( K m The kinetics is 11.22 nM; when TMB is used as a variable substrate, the reaction kinetics curve is as follows: Figure 2 As shown in (f), the corresponding V max It is 138.3 nM / s. K m The value is 0.063 nM. These parameters indicate that the maifanite nanozyme can effectively interact with the substrate and maintain a stable catalytic reaction rate.

[0037] Furthermore, free radicals in the reaction system were detected using electron spin resonance (ESR) technology, and the results were as follows: Figure 2 As shown in (g), a hydroxyl radical (•OH) signal can be detected in the reaction system containing maifanite nanozymes, indicating that the nanozyme can induce the generation of active intermediates during the catalytic process. The process by which maifanite nanozymes catalyze the decomposition of hydrogen peroxide and generate hydroxyl radicals is as follows: Figure 2 As shown in (a). Further testing was conducted to investigate the relationship between the amount of maifanite nanozyme and the catalytic response, and the results are as follows. Figure 2 As shown in (h), with the increase of the amount of maifanite nanozyme, the absorption signal of the reaction system gradually increases, showing a dose-related trend.

[0038] The test results above show that the maifanite nanozyme of the present invention exhibits stable peroxidase-like catalytic characteristics in a specific reaction system and has good adaptability under different pH and temperature conditions, providing a catalytic basis for its participation in redox regulation in plant systems.

[0039] The maifanite nanozyme prepared in Example 1 of this invention was dispersed in deionized water to obtain a maifanite nanozyme dispersion of 0.53 mg / mL.

[0040] Wild tobacco (Nicotiana benthamiana) was selected as the research object. The 0.53 mg / mL maifanite nanoenzyme dispersion prepared in Example 1 was applied via foliar spraying. Untreated plants served as the control group. The tissue distribution and plant growth traits after application of the maifanite nanoenzyme dispersion were examined. The results are as follows: Figure 3 As shown in (a), firstly, hyperspectral images of maifanite nanozymes were acquired and a corresponding spectral feature library was established. Subsequently, the spectral feature library was applied to hyperspectral imaging analysis of tobacco leaf cells.

[0041] Depend on Figure 3(a) The results showed that regions with characteristic scattering signals could be identified in the samples treated with maifanite nanozymes, while the same scattering characteristics were not observed in the control group samples. Further imaging analysis showed that the scattering signals were distributed in a point-like form at the cell edges and intercellular spaces, and magnified images revealed that the signals were mainly located at the interface between the cell wall and the extracellular space. These results indicate that maifanite nanozymes can enter or attach to plant leaf tissues after application, and are mainly distributed in the extracellular or pericellular regions.

[0042] To further verify the distribution of maifanite nanozymes within plants, inductively coupled plasma atomic emission spectrometry (ICP) was used to determine the content of silicon (Si) and aluminum (Al) in different tissues of tobacco plants (silicon and aluminum are the main elemental components of maifanite nanozymes). The results are as follows: Figure 3 (b) and Figure 3 As shown in (c).

[0043] Depend on Figure 3 (b) and Figure 3 (c) It was found that, compared with the control group, the content of silicon and aluminum in the leaf and stem tissues of tobacco plants treated with maifanite nanoenzymes was increased, indicating that silicon and aluminum were effectively distributed and transferred within the plant. In contrast, such as Figure 3 As shown in (d), the silicon and aluminum content in the roots showed a decreasing trend compared to the control group. These results indicate that under foliar application conditions, silicon and aluminum elements in maifanite nanoenzymes can directly enter the aboveground tissues, thereby reducing dependence on root absorption pathways. At the same time, it may trigger the regulation process of element allocation within the plant, leading to a decrease in the relative content of related elements in the roots.

[0044] The growth traits of tobacco plants were observed and statistically analyzed at the whole-plant level. The results are as follows: Figure 3 As shown in (e), the tobacco plants treated with maifanite nanoenzymes exhibited better overall growth than the control group. Further quantitative analysis revealed an increase in both the number of leaves and branches in the treatment group. Figure 3 (f)–(g)). Furthermore, growth indicators such as plant height, leaf width, stem diameter, and chlorophyll content all showed an upward trend. Figure 3 (h)–(k)). It can be seen that the maifanite nanoenzyme of the present invention can be effectively distributed in the leaf and stem tissues of plants after application, and promotes the growth and development of plants at the overall level.

[0045] Given that the maifanite nanozyme of this invention possesses endogenous peroxidase-like activity, its effect on redox state in plant cells was further investigated. A DCFH-DA fluorescently labeled reactive oxygen species (ROS) probe, combined with confocal microscopy, was used to observe the ROS levels in treated plant cells. Figure 4 As shown in (a), compared with the control group, enhanced ROS fluorescence signal was detected in tobacco cells treated with maifanite nanozymes, but no obvious abnormal changes were observed in the overall cell morphology, indicating that the cell structure remained intact under the treatment conditions. This result shows that maifanite nanozyme treatment can induce changes in intracellular redox activity without causing significant cell damage.

[0046] The activities of antioxidant enzymes related to redox regulation in plant leaf tissues were further investigated. The results are as follows: Figure 4 (b) and Figure 4 As shown in (c), after application of maifanite nanozymes, the activities of superoxide dismutase (SOD) and catalase (CAT) in leaf tissues were both higher than those in the control group. Simultaneously, the content of malondialdehyde (MDA), an indicator of lipid peroxidation levels, was measured. Figure 4 (d) The results showed no significant difference between the treatment group and the control group. These results indicate that maifanite nanozyme treatment, while increasing antioxidant enzyme activity, did not cause an increase in lipid peroxidation levels.

[0047] To further analyze the molecular basis of the regulation of plant redox state by maifanite nanozymes and its impact on growth, transcriptome sequencing analysis was performed on leaf samples from the maifanite nanozyme treatment group and the control group. Differential expression analysis showed that, under statistical threshold conditions, multiple genes exhibited altered expression levels in the treatment group, indicating a significant transcriptional response in plant cells. Functional enrichment analysis of the differentially expressed genes revealed a high enrichment level of gene sets related to functional categories such as oxidative stress response, defense response, and extracellular processes in the treatment group. Further pathway enrichment analysis showed a high enrichment level of photosynthesis-related pathways, especially those related to photosynthetic antenna proteins, in the treatment group, indicating responsive changes in light capture-related processes. Furthermore, multiple pathways related to redox regulation and metabolic processes were also enriched in the treatment group, including porphyrin metabolism, glutathione metabolism, sulfur metabolism, and vitamin B6 metabolism pathways. Simultaneously, pathways related to the synthesis of phenylpropanes, flavonoids, and terpenoids, as well as pathways related to plant-pathogen interactions, were also detected, reflecting a synergistic response in plants at metabolic and defense levels. For example... Figure 4As shown in (e)–(m), gene set enrichment analysis (GSEA) further revealed that multiple functional gene sets related to redox regulation, energy metabolism, and cellular homeostasis maintenance exhibited a positive enrichment trend in the maifanite nanozyme treatment group. Among them, gene sets related to oxidative stress response, antioxidant activity, peroxidase-related functions, and heme binding processes showed significant enrichment characteristics. Figure 4 (e)–(j)). Meanwhile, metabolic pathways related to carbohydrate catabolism, sulfur-containing amino acid metabolism, and isoprene biosynthesis were also activated in the treatment group. Figure 4 (k) – (m)).

[0048] The results above show that the maifanite nanozyme of the present invention can induce a regulatory response of redox-related processes in plant cells, accompanied by synergistic changes in multiple metabolic and energy utilization pathways, thus providing molecular-level support for its application in plant growth, material accumulation and quality improvement.

[0049] To further analyze the effects of the maifanite nanoenzyme treatment of this invention on the metabolic level in plants, non-targeted metabolomics analysis was conducted using tobacco leaf tissue as the research object. For example... Figure 5 As shown in (a), principal component analysis (PCA) was performed on the metabolic data of the treatment and control groups. The results showed that the two groups were significantly different in the first and second principal component directions, indicating that the overall metabolic characteristics of plant leaves changed after the application of maifanite nanozymes. In this study, a total of 224 metabolite characteristics were detected and quantitatively analyzed. Figure 5 As shown in (b), compared with the control group, the abundance of various metabolites changed in the maifanite nanozyme treatment group, with some metabolites showing an upregulation trend and others showing a downregulation trend. After classifying the samples and differentially expressed metabolites through hierarchical cluster analysis, it was found that the treatment group and the control group had different metabolic characteristics in terms of metabolite composition and abundance distribution.

[0050] Further screening of differentially expressed metabolites was performed using projection importance analysis (VIP), and the results are as follows: Figure 5 As shown in (c), amino acids and their derivatives, alkaloids, flavonoids, and lipid-related metabolites contributed significantly to differentiating the treatment and control groups, indicating that plants underwent responsive changes at both the primary and secondary metabolic levels. Pathway enrichment analysis based on differential metabolites is shown below. Figure 5As shown in (d), multiple metabolic pathways related to redox regulation and nutrient metabolism were affected in the treatment group. Among them, pathways related to amino acid metabolism (including arginine and proline metabolism, lysine degradation, alanine, aspartic acid, and glutamate metabolism), carbon metabolism, glutathione metabolism, and porphyrin metabolism all showed an enrichment trend. Furthermore, metabolic processes related to flavonoid biosynthesis and phosphatidylinositol signaling pathways were also detected, reflecting changes in antioxidant-related metabolism and signal regulation in plants. Figure 5 As shown in (e), visualization analysis of representative differential metabolites using radar charts revealed synergistic changes in various compounds related to redox regulation, amino acid derivatives, and lipid metabolism in the maifanite nanozyme treatment group. Among these, the abundance of phosphatidylinositol-related lipids showed significant changes, indicating a marked adjustment in membrane lipids and related signaling molecules. Furthermore, the relative abundance of modified nucleoside metabolites such as 5-methyluridine and isoxanthopterin increased, reflecting changes in nucleotide metabolism-related processes. Simultaneously, the fatty acid-derived amide compound N-methylundec-10-enamide also showed an upregulation trend in the treatment group. Among polypeptide metabolites, different peptides exhibited changes in different directions, reflecting adjustments in protein metabolism and amino acid reuse processes. On the other hand, the abundance of some stress-response-related metabolites showed a decreasing trend in the treatment group, indicating corresponding changes in the plant's metabolic state.

[0051] The above metabolomics analysis results show that the treatment of maifanite nanoenzymes in this invention can induce systematic changes in the metabolic profile of plant leaves, involving multiple aspects such as amino acid metabolism, lipid metabolism, antioxidant-related metabolism and signal molecule synthesis, providing experimental evidence at the metabolic level for its application in plant growth, material accumulation and quality improvement.

[0052] The maifanite nanozyme prepared in Example 1 of this invention was dispersed in deionized water to obtain a maifanite nanozyme dispersion of 0.53 mg / mL.

[0053] Wheat was selected as the target crop. Throughout its growth period, the maifanite nanoenzyme dispersion (0.53 mg / mL) prepared in Example 1 was applied via foliar spraying. The control group received only an equal volume of deionized water. All treatment groups underwent field management under the same cultivation conditions. After crop maturity, the growth traits and grain yield of the wheat plants were measured. Figure 6 As shown in (a), the morphology of wheat ears was observed during the grain-filling stage. Compared with the control treatment, the wheat plants treated with maifanite nanoenzymes showed better ear development and different grain filling.

[0054] Further statistical analysis of the output composition yielded the following results: Figure 6As shown in (b), under non-stress growth conditions, the total wheat grain yield in the plots treated with maifanite nanozymes showed an increasing trend compared to the control plots, with a yield increase of 18.74% per plot. During the above yield increase process, no abnormal plant morphology or obvious stress-related phenotypes were observed.

[0055] Further testing of the nutritional quality indicators of wheat grains yielded the following results: Figure 6 As shown in (c), compared with the control group, the total protein content in wheat grains treated with maifanite nanoenzymes increased from 159.6 ± 1.513 g / kg to 186.5 ± 5.329 g / kg, indicating a change in grain protein accumulation. Simultaneously, quantitative analysis of the free amino acid composition in wheat grains revealed the detection of 18 free amino acids. Figure 6 (d) and Figure 6 As shown in (e), compared with the control group, the content of various free amino acids in the grains of the treated group changed, including essential amino acids such as leucine, phenylalanine, valine, isoleucine, threonine, lysine, methionine, and histidine, as well as non-essential amino acids such as glutamic acid, proline, aspartic acid, serine, glycine, alanine, and cysteine, showing an overall decreasing trend. In contrast, the tryptophan content showed an increasing trend in the maifanite nanoenzyme treatment group. Combined with the results of grain yield and total protein content detection, it can be seen that after applying maifanite nanoenzymes, the composition and distribution of free amino acids in wheat grains changed, reflecting the adjustment of nitrogen utilization and protein synthesis-related metabolic processes.

[0056] The results of the field trials above show that the maifanite nanoenzyme of this invention can have a positive impact on crop yield and grain quality under different environmental conditions in actual agricultural production, demonstrating its application potential in agricultural production increase and quality control.

Claims

1. A maifanite nanoenzyme, characterized in that, It is prepared by modifying maifanite through mechanical activation. The maifanite nanozyme has the mineral composition, nanoscale size characteristics and peroxidase-like activity of maifanite.

2. The maifanite nanoenzyme as described in claim 1, characterized in that, The maifanite nanozyme has a nanoscale particle morphology with a particle size of 120-500 nm.

3. The method for preparing the maifanite nanozyme as described in claim 1 or 2, characterized in that, Includes the following steps: (1) Maifan stone pretreatment; (2) Homogenization: The maifan stone pretreated in step (1) is physically crushed and ground to obtain micron-sized maifan stone powder; (3) Nano-sizing treatment: The micron-sized maifanite powder from step (2) is nano-sized to obtain maifanite nanoenzymes.

4. The method for preparing maifanite nanozymes as described in claim 3, characterized in that, In step (3), the nano-sizing method adopts high-energy ball milling, ultrasonic exfoliation or liquid phase shearing exfoliation.

5. The method for preparing maifanite nanozymes as described in claim 4, characterized in that, In step (2), physical crushing and grinding are carried out using an impact-type ultrafine pulverizer. The working conditions are: running continuously for 3-6 times at a speed of 2000-3000 rpm, with a total running time of 20-30 min. In step (3), the nano-processing method adopts high-energy ball milling, specifically: the micron-sized maifanite powder from step (2) is placed in a high-energy nano-impact mill, the mass ratio of the ball milling medium to the maifanite powder is about (2-6):1, the filling rate of the ball milling medium is 25%-50%, and the ball milling is carried out for 40-60 min at a vibration frequency of 15-50 Hz and a temperature of 15-20 ℃, and then sieved to obtain maifanite nanoenzyme.

6. The method for preparing maifanite nanozymes as described in claim 3, characterized in that, In step (1), the pretreatment method for maifan stone is to wash the maifan stone with water 1-3 times and then dry it. In step (3), the ball milling media is composed of steel balls of different particle sizes, wherein steel balls with a diameter of 1 cm account for 50-60% of the total steel ball mass, steel balls with a diameter of 0.6 cm account for 25-30% of the total steel ball mass, and steel balls with a diameter of 0.4 cm account for 15-20% of the total steel ball mass.

7. The application of the maifanite nanoenzyme as described in claim 1 or 2 in the preparation of agricultural fertilizers, plant growth regulators or soil conditioners.

8. The application as described in claim 7, characterized in that, The maifanite nanoenzyme, as a component of plant growth regulators, regulates the redox homeostasis within plant cells, causing changes in the metabolic spectrum of plant leaves and promoting root development.

9. The application as described in claim 8, characterized in that, The plant in question is either wheat or tobacco.

Images