Eugenol-nanometer enzyme-sodium alginate hydrogel spray for preventing and treating gray mold of ginseng as well as preparation method and application of eugenol-nanometer enzyme-sodium alginate hydrogel spray

Abstract

The invention discloses an eugenol-nanometer enzyme-sodium alginate hydrogel spray for preventing and treating gray mold of ginseng as well as a preparation method and application of the eugenol-nanometer enzyme-sodium alginate hydrogel spray, and relates to the technical field of pesticides. The preparation method comprises the following steps: carrying out a coupling reaction on sodium alginate and dopamine hydrochloride to prepare a DA-SA hydrogel precursor; dissolving the DA-SA hydrogel precursor in water to obtain a precursor solution; and dispersing the eugenol at nano-enzyme in the precursor solution to obtain a nano-enzyme hydrogel precursor mixed suspension, namely the eugenol at nano-enzyme-sodium alginate hydrogel spray. The eugenol-nanometer enzyme-sodium alginate hydrogel spray disclosed by the invention not only has a good slow-release characteristic, but also enhances the rain wash resistance; meanwhile, the loaded pesticide components are natural and green and harmless to human, environment and livestock, and the good biocompatibility of the nano-enzyme ensures the safety and effectiveness of the hydrogel composite pesticide in pesticide technology application.

Description

Technical Field

[0001] The present invention relates to the technical field of pesticides, and in particular to an eugenol@nanozyme-sodium alginate hydrogel spray for preventing and treating Botrytis cinerea of ginseng, and a preparation method and application thereof. Background Art

[0002] Botrytis cinerea belongs to the pathogen of Botrytis cinerea, and is a highly destructive necrotrophic phytopathogenic fungus. It can infect a variety of plants, including tomatoes, cucumbers, soybeans, ginseng, etc. A series of virulence factors will be produced during the infection process of this bacterium, including oxalic acid toxin, cell wall degrading enzymes, ATP-binding cassette transporters, pectin methyl esterase, etc. A large amount of oxalic acid secreted therein will destroy the pH value of the infected part of the host plant, and the acidic environment increases the activities of cell wall degrading enzymes and enzymes promoting pathogen colonization, thereby causing symptoms similar to dark water soaking in the infected part, that is, Botrytis cinerea. At present, the commonly used prevention and control strategies for Botrytis cinerea of ginseng in the market are still the use of synthetic fungicides. However, the large-scale use of synthetic fungicides has certain negative impacts, including the destruction of the ecological balance, the generation of resistance in fungal pathogens, and the harm of pesticide residues to human health. These problems have prompted people to develop plant-derived fungicides with high efficiency, safe and easy degradation, low resistance, and high selectivity.

[0003] The active ingredient of plant-derived fungicides is natural compounds, which are relatively easy to be rapidly decomposed into non-toxic substances in the environment through photolysis, hydrolysis and microbial action after application, can reduce soil and water pollution, and due to the diversification of its components, it is difficult for pathogens to develop drug resistance. As a plant-derived fungicide, eugenol has a variety of biological activities and has been used for a long time to control plant pathogens and pests. However, eugenol has problems such as strong volatility and poor solubility, resulting in poor persistence, making its bactericidal effect far from comparable to that of traditional chemical pesticides. And in the actual application process, eugenol is significantly limited due to its poor resistance to rain erosion, manifested as rapid leaching and shortened retention period under rainfall conditions. Therefore, it is necessary to develop a new dosage form of eugenol fungicide to improve its bactericidal effect. Summary of the Invention

[0004] The purpose of the present invention is to provide an eugenol@nanozyme-sodium alginate hydrogel spray for preventing and treating Botrytis cinerea of ginseng, and a preparation method and application thereof, so as to solve the problems existing in the above-mentioned prior art. The eugenol@nanozyme-sodium alginate hydrogel spray of the present invention not only has good sustained-release characteristics, but also enhances the resistance to rain erosion; at the same time, the loaded pesticide components are natural and green, harmless to humans, the environment and livestock, and the good biocompatibility of the nanozyme ensures the safety and effectiveness of the hydrogel composite pesticide in the application of pesticide technology.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a preparation method of eugenol@nanozyme for preventing and treating gray mold of ginseng, comprising the following steps:

[0007] Calcine ZIF-67 to obtain nanozyme;

[0008] Mix and react the nanozyme and eugenol in a solvent to prepare the eugenol@nanozyme.

[0009] Further, the temperature of the calcination treatment is 600-800°C and the time is 3 h.

[0010] Further, the mass ratio of the nanozyme to the eugenol is 1:4-6.

[0011] Further, the solvent is dimethyl sulfoxide.

[0012] The present invention also provides an eugenol@nanozyme prepared by the above preparation method.

[0013] The present invention also provides a preparation method of an eugenol@nanozyme-sodium alginate hydrogel spray for preventing and treating gray mold of ginseng, comprising the following steps:

[0014] Perform a coupling reaction on sodium alginate and dopamine hydrochloride to prepare a DA-SA hydrogel precursor;

[0015] Dissolve the DA-SA hydrogel precursor in water to obtain a precursor solution;

[0016] Disperse the above eugenol@nanozyme in the precursor solution to obtain a nanozyme hydrogel precursor mixed suspension, which is the eugenol@nanozyme-sodium alginate hydrogel spray.

[0017] The present invention also provides an eugenol@nanozyme-sodium alginate hydrogel spray prepared by the above preparation method.

[0018] The present invention also provides the application of the above eugenol@nanozyme in preventing and treating gray mold of ginseng.

[0019] The present invention also provides the application of the above eugenol@nanozyme-sodium alginate hydrogel spray in preventing and treating gray mold of ginseng.

[0020] The present invention also provides a method for preventing and treating gray mold of ginseng, comprising the step of sequentially spraying the above eugenol@nanozyme-sodium alginate hydrogel spray and an aqueous CaCl2 solution on ginseng plants.

[0021] The present invention discloses the following technical effects:

[0022] The eugenol@nanozyme prepared by the present invention uses a biomimetic nanozyme to load the plant-derived fungicide eugenol. The nanozyme has high catalytic performance and simulates the catalytic function of peroxidase-like (OXD). Under acidic conditions at the disease site, it can catalyze O2 to produce superoxide anions to play a synergistic antifungal role. Under the action of the nanozyme, the problems of strong volatility and slow drug efficacy of eugenol can be effectively solved. At the same time, the antifungal effect can be further improved while reducing the amount of pesticide used, thus avoiding the problem of drug resistance caused by excessive use of antibacterial drugs.

[0023] The present invention loads eugenol@nanozyme into a hydrogel matrix to prepare an eugenol@nanozyme-sodium alginate hydrogel spray. This dual-loaded nanozyme-hydrogel hybrid system synergistically integrates the slow-release characteristics of nanoparticles and the adhesion characteristics of hydrogels, providing a robust delivery platform for optimizing the rain resistance of plant-derived fungicides and nano-pesticides.

[0024] The eugenol@nanozyme-sodium alginate hydrogel spray of the present invention not only has good slow-release characteristics but also enhances the rain resistance performance; at the same time, the loaded pesticide components are natural and green, harmless to humans, the environment, and livestock, and the good biocompatibility of the nanozyme ensures the safety and effectiveness of the hydrogel composite pesticide in the application of pesticide technology. Brief Description of the Drawings

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0026] Figure 1 Scanning electron microscope images of eugenol@nanozyme (A) and eugenol@nanozyme-sodium alginate hydrogel (B);

[0027] Figure 2 Particle size statistical charts of nanozyme (A) and eugenol@nanozyme (B);

[0028] Figure 3 Infrared spectra of eugenol, nanozyme, eugenol@nanozyme, sodium alginate hydrogel, and eugenol@nanozyme-sodium alginate hydrogel; EU represents eugenol; CoSA represents nanozyme; EU@CoSA represents eugenol@nanozyme; DA-SA represents sodium alginate hydrogel; DA-SA+EU@CoSA represents eugenol@nanozyme-sodium alginate hydrogel;

[0029] Figure 4Thermogravimetric analysis diagrams of eugenol, nanozyme, and eugenol@nanozyme; EU represents eugenol; CoSA represents nanozyme; EU@CoSA represents eugenol@nanozyme;

[0030] Figure 5 Water contact angle analysis diagrams of eugenol, eugenol@nanozyme, and eugenol@nanozyme-sodium alginate hydrogel; EU represents eugenol; EU@CoSA represents eugenol@nanozyme; DA-SA+EU@CoSA represents eugenol@nanozyme-sodium alginate hydrogel;

[0031] Figure 6 Statistical graphs of leaf liquid holding capacity of eugenol, eugenol@nanozyme, and eugenol@nanozyme-sodium alginate hydrogel; EU represents eugenol; EU@CoSA represents eugenol@nanozyme; DA-SA+EU@CoSA represents eugenol@nanozyme-sodium alginate hydrogel;

[0032] Figure 7 Graph of the OXD-like enzyme activity of eugenol@nanozyme;

[0033] Figure 8 In vitro drug release graph of eugenol@nanozyme-sodium alginate hydrogel spray;

[0034] Figure 9 Graph of the biosafety detection results of nanozyme;

[0035] Figure 10 Graph of the in vitro antifungal activity of different pesticides; among them, A is the graph of the in vitro antifungal activity of the blank control group, B is the graph of the in vitro antifungal activity of acetone, C is the graph of the in vitro antifungal activity of eugenol, D is the graph of the in vitro antifungal activity of nanozyme, E is the graph of the in vitro antifungal activity of eugenol@nanozyme, and F is the graph of the in vitro antifungal activity of eugenol@nanozyme-sodium alginate hydrogel spray;

[0036] Figure 11 Graph of the antifungal activity of different pesticides on detached leaves; among them, A is the graph of the antifungal activity of the blank control group on detached leaves, B is the graph of the antifungal activity of eugenol on detached leaves, C is the graph of the antifungal activity of nanozyme on detached leaves; D is the graph of the antifungal activity of eugenol@nanozyme-sodium alginate hydrogel spray on detached leaves. Detailed implementation manners

[0037] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0038] It should be understood that the terms described in the present invention are only for describing particular embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0039] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0040] Without departing from the scope or spirit of the present invention, various improvements and variations can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0041] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0042] In the present invention, EU represents eugenol; CoSA represents nanozyme; EU@CoSA represents eugenol@nanozyme; DA-SA represents sodium alginate hydrogel; DA-SA+EU@CoSA represents eugenol@nanozyme-sodium alginate hydrogel.

[0043] Based on the characteristic that Botrytis cinerea infects plant parts to produce oxalic acid, leading to a decrease in pH, the present invention designs an eugenol@nanozyme-sodium alginate hydrogel spray. The bionic nanozyme is loaded with the plant-derived fungicide eugenol. The nanozyme has high catalytic performance and simulates the catalytic function of OXD enzyme. Under acidic conditions at the disease site, it can catalyze O2 to produce superoxide anions to play a synergistic antifungal role. Under the action of the nanozyme, it can not only effectively solve the problems of strong volatility and slow drug efficacy of eugenol, but also further improve the antifungal effect while reducing the amount of pesticide used, thus avoiding the problem of drug resistance caused by excessive use of antibacterial drugs. At the same time, the eugenol nanozyme-hydrogel composite pesticide adopts a hierarchical encapsulation strategy. First, the bionic nanozyme is loaded with the plant-derived fungicide eugenol, and then the eugenol@nanozyme is loaded into the hydrogel matrix. This dual-loaded nanozyme-hydrogel hybrid system synergistically integrates the slow-release characteristics of nanoparticles and the adhesion and anti-swelling characteristics of hydrogels, providing a robust delivery platform for optimizing the rain resistance of plant-derived fungicides and nano-pesticides.

[0044] In the examples of the present invention, the room temperature is uniformly calculated as 25±2°C.

[0045] In the examples of the present invention, the epidermal cells are purchased from Fuheng Biotechnology Co., Ltd.; the fetal bovine serum is CLARKFB15015 high-quality fetal bovine serum; penicillin / streptomycin is purchased from Sevier Biotechnology Co., Ltd.; the DMEM medium is purchased from gibco DMEM high-glucose liquid medium; eugenol is purchased from Shanghai Macklin Biochemical Co., Ltd.

[0046] Example 1

[0047] A preparation method of an eugenol@nanozyme-sodium alginate hydrogel spray:

[0048] S1. Mix 2-methylimidazole and methanol according to a molar volume ratio of 7 mmol:15 mL to obtain a 2-methylimidazole solution; mix cobalt nitrate hexahydrate and methanol according to a molar volume ratio of 0.9 mmol:15 mL to obtain a cobalt nitrate solution. Mix the cobalt nitrate solution and the 2-methylimidazole solution in equal volume and stir for reaction. The stirring time is 30 min, the stirring rate is 500 rpm, centrifuge at 12000 rpm for 10 min to take the precipitate, wash it repeatedly with anhydrous methanol, and then place the solid product in a vacuum dryer at 60°C for 12 h to obtain ZIF-67 powder.

[0049] S2. Place the ZIF-67 powder obtained in step S1 in a tube furnace, heat it to 600°C at a rate of 5°C / min in an argon atmosphere, and calcine for 3 h to obtain nanozyme.

[0050] S3. Disperse the nanozyme obtained in step S2 in dimethyl sulfoxide (DMSO), and perform ultrasonic treatment for 10 min (power 100 W) to eliminate aggregation. Then add eugenol and mix and stir at room temperature (25 °C) (500 rpm) for 24 h to load eugenol on the nanozyme, where the mass ratio of nanozyme to eugenol is 1:5, and the mass-volume ratio of eugenol to DMSO is 500 mg:10 mL. Wash three times with ethanol, centrifuge at 8000 rpm for 10 min to obtain the precipitate, and vacuum dry at 60 °C for 6 h to obtain eugenol@nanozyme.

[0051] S4. Dissolve 2 g of sodium alginate in deionized water to prepare a 1 wt% sodium alginate solution. Sequentially add 1.725 g of EDC·HCl and 1.036 g of NHS to the solution. After reacting at room temperature for 3 h, add 1.7 g of dopamine hydrochloride, adjust the pH to 4.5 with 2 M HCl, and react at room temperature under argon for 24 h. Then place it in a 3500 KD dialysis bag and dialyze with deionized water for 3 d, and freeze-dry to obtain a solid. Then dissolve it in deionized water, adjust the pH to 8.5 with 2 M NaOH, stir at room temperature for 5 h, place it in a 3500 KD dialysis bag and dialyze with deionized water for 1 d, and freeze-dry to obtain the DA-SA hydrogel precursor. The process parameters for the freeze-drying process are: pre-freeze at -80 °C for 180 min, vacuum freeze-dry at -60 °C, freeze-dry for 48 h, and the vacuum degree is 1 Pa.

[0052] S5. Dissolve the DA-SA hydrogel precursor obtained in step S4 in deionized water to obtain a 4 mg / mL precursor solution. Disperse the eugenol@nanozyme obtained in step S3 in the DA-SA hydrogel precursor solution to obtain a nanozyme hydrogel precursor mixed suspension, and combine it with a 2 wt% CaCl2 aqueous solution. Spray in sequence, first spray the nanozyme hydrogel precursor mixed suspension, and then spray the 2 wt% CaCl2 aqueous solution to obtain the eugenol@nanozyme-sodium alginate hydrogel.

[0053] Example 2

[0054] A preparation method of an eugenol@nanozyme-sodium alginate hydrogel spray:

[0055] S1. Mix 2-methylimidazole and methanol according to a molar volume ratio of 6 mmol:15 mL to obtain a 2-methylimidazole solution; mix cobalt nitrate hexahydrate and methanol according to a molar volume ratio of 0.8 mmol:15 mL to obtain a cobalt nitrate solution. Mix the cobalt nitrate solution and the 2-methylimidazole solution in equal volume and stir and react. The stirring time is 45 min, the stirring rate is 400 rpm, centrifuge at 12000 rpm for 10 min to obtain the precipitate, and dry the solid product at 60 °C for 18 h to obtain ZIF-67 powder.

[0056] S2. Place the ZIF-67 powder obtained in step S1 into a tubular furnace, heat it to 700 °C at a rate of 5 °C / min in an argon atmosphere, and calcine for 3 h to obtain the nanozyme.

[0057] S3. Disperse the nanozyme obtained in step S2 in DMSO, and perform ultrasonic treatment for 10 min (power 100 W) to eliminate agglomeration. Then add eugenol and mix and stir at room temperature (25 °C, 500 rpm) for 36 h to load eugenol onto the nanozyme. The mass ratio of nanozyme to eugenol is 1:4, and the mass-volume ratio of eugenol to DMSO is 400 mg:10 mL. Wash three times with ethanol, centrifuge at 8000 rpm for 10 min to collect the precipitate, and vacuum dry at 60 °C for 6 h to obtain eugenol@nanozyme.

[0058] S4. Dissolve 2 g of sodium alginate in deionized water to prepare a 1 wt% sodium alginate solution. Sequentially add 1.725 g of EDC·HCl and 1.036 g of N-hydroxysuccinimide to the solution, react at room temperature for 3 h, then add 1.7 g of dopamine hydrochloride, adjust the pH to 4.5 with 2 M HCl, pass argon and react at room temperature for 24 h, then place it in a 3500 KD dialysis bag and dialyze with deionized water for 4 d. Freeze-dry to obtain a solid, dissolve it in deionized water again, adjust the pH to 8.5 with 2 M NaOH, stir at room temperature for 5 h, place it in a 3500 KD dialysis bag and dialyze with deionized water for 1 d, and then freeze-dry to obtain the DA-SA hydrogel precursor. The process parameters for the freeze-drying process are: pre-freeze at -80 °C for 180 min, vacuum freeze-dry at -60 °C, freeze-dry for 48 h, and the vacuum degree is 1 Pa.

[0059] S5. Dissolve the DA-SA hydrogel precursor obtained in step S4 in deionized water to obtain a 4 mg / mL precursor solution. Disperse the eugenol@nanozyme obtained in step S3 in the DA-SA hydrogel precursor solution to obtain a nanozyme hydrogel precursor mixed suspension. Combine it with a 2 wt% CaCl2 aqueous solution and spray in sequence. First, spray the nanozyme hydrogel precursor mixed suspension, and then spray the 2 wt% CaCl2 aqueous solution to obtain the eugenol@nanozyme-sodium alginate hydrogel.

[0060] Example 3

[0061] A preparation method of a eugenol@nanozyme-hydrogel composite pesticide:

[0062] S1. Mix 2-methylimidazole with methanol at a molar volume ratio of 8 mmol:15 mL to obtain a 2-methylimidazole solution; mix cobalt nitrate hexahydrate with methanol at a molar volume ratio of 1 mmol:15 mL to obtain a cobalt nitrate solution. Mix the cobalt nitrate solution and the 2-methylimidazole solution in equal volumes and stir for reaction. The stirring time is 60 min, the stirring rate is 600 rpm, centrifuge at 12000 rpm for 10 min to collect the precipitate, and place the solid product in a vacuum dryer at 60 °C for 24 h to obtain ZIF-67 powder.

[0063] S2. Place the ZIF-67 powder obtained in step S1 in a tube furnace, heat it to 800 °C at a rate of 5 °C / min in an argon atmosphere, and calcine for 3 h to obtain a nanozyme.

[0064] S3. Disperse the nanozyme in DMSO, perform ultrasonic treatment for 10 min (power 100 W) to eliminate agglomeration, add eugenol, and mix and stir at room temperature (25 °C) (500 rpm) for 36 h to load eugenol onto the nanozyme. The mass ratio of the nanozyme to eugenol is 1:6, and the mass-volume ratio of eugenol to DMSO is 600 mg:10 mL; wash three times with ethanol, centrifuge at 8000 rpm for 10 min to collect the precipitate, and vacuum dry at 60 °C for 6 h to obtain eugenol@nanozyme.

[0065] S4. Dissolve 2 g of sodium alginate in deionized water to prepare a 1 wt% sodium alginate solution. Sequentially add 1.725 g of EDC·HCl and 1.036 g of N-hydroxysuccinimide to the solution, react at room temperature for 3 h, then add 1.7 g of dopamine hydrochloride, adjust the pH to 4.5 with 2 M HCl, react at room temperature while purging with argon for 24 h, place it in a 3500 KD dialysis bag and dialyze with deionized water for 5 d, freeze-dry to obtain a solid, then dissolve it in deionized water, adjust the pH to 8.5 with 2 M NaOH, stir at room temperature for 5 h, place it in a 3500 KD dialysis bag and dialyze with deionized water for 1 d, and freeze-dry to obtain a DA-SA hydrogel precursor. The process parameters for the freeze-drying process are: pre-freeze at -80 °C for 180 min, vacuum freeze-dry at -60 °C, freeze-dry for 48 h, and the vacuum degree is 1 Pa.

[0066] S5. Dissolve the DA-SA hydrogel precursor obtained in step S4 in deionized water to obtain a 4 mg / mL precursor solution. Disperse the eugenol@nanozyme obtained in step S3 in the DA-SA hydrogel precursor solution to obtain a nanozyme hydrogel precursor mixed suspension, and use it in combination with a 2 wt% aqueous CaCl2 solution. Spray in sequence, first spray the nanozyme hydrogel precursor mixed suspension, and then spray the 2 wt% aqueous CaCl2 solution to obtain eugenol@nanozyme-sodium alginate hydrogel.

[0067] Example 4

[0068] The following performance tests were carried out on the eugenol@nanozyme-sodium alginate hydrogel prepared in Example 1:

[0069] 1. Structural characterization

[0070] Figure 1 are the scanning electron microscope images of eugenol@nanozyme and eugenol@nanozyme-sodium alginate hydrogel. As can be seen from Figure 1 A in it, eugenol@nanozyme presents a rhombic dodecahedron, and as can be seen from Figure 1 B in it, eugenol@nanozyme is evenly distributed in the hydrogel.

[0071] Figure 2 are the particle size statistical data graphs of nanozyme (A) and eugenol@nanozyme (B). The results show that the average particle size of nanozyme is 307.13±34.14 nm, and the average particle size of eugenol@nanozyme is 309.58±32.25 nm.

[0072] Qualitative analysis was carried out on eugenol, nanozyme, eugenol@nanozyme, sodium alginate hydrogel and eugenol@nanozyme-sodium alginate hydrogel samples using Fourier transform infrared spectroscopy. The results are shown in Figure 3 . In the eugenol sample, the characteristic peaks appearing at 3200 - 3400 cm -1 are attributed to the stretching vibration of the O-H bond, and the characteristic peaks appearing at 3000 - 3100 cm -1 are attributed to the stretching vibrations of aromatic C-H bonds and allylic C-H bonds. The characteristic peaks of eugenol at 3200 - 3400 cm -1 and 3000 - 3100 cm -1 can also be observed in eugenol@nanozyme and eugenol@nanozyme-sodium alginate hydrogel, which further confirms that eugenol is encapsulated in the nanozyme.

[0073] Qualitative analysis was carried out on eugenol, nanozyme and eugenol@nanozyme samples using a thermogravimetric analyzer. The results are shown in Figure 4 . The thermogravimetric analysis curve shows that when approaching 200 °C, the weight loss of eugenol is about 100%, while the weight losses of nanozyme and eugenol@nanozyme between 25 °C and 800 °C are about 19.15 and 39.33 respectively. It can be seen from this that the loading rate of eugenol in eugenol@nanozyme is 20.18%.

[0074] The adhesion ability of pesticides to plant leaves after spraying is an important factor affecting the utilization rate of pesticides. The present invention studied the adhesion characteristics of eugenol@nanozyme-hydrogel composite pesticides on ginseng leaves. Figures 5 - 6 are the water contact angle analysis diagram and leaf liquid holding capacity diagram of the eugenol@nanozyme-sodium alginate hydrogel spray. The droplet contact angles formed by different components on ginseng leaves are as shown in Figure 5As shown. Compared with H2O, eugenol, and eugenol@nanozyme, the eugenol@nanozyme hydrogel precursor mixed suspension has a smaller droplet contact angle on ginseng leaves. The wettability of the formulation was evaluated by measuring the liquid holding capacity (LHC), which directly affects the accumulation of active ingredients on the leaves. Figure 6 The liquid holding capacities of eugenol, eugenol@nanozyme, and the eugenol@nanozyme-hydrogel precursor mixed suspension are shown. Before the experiment, the ginseng plant leaves were gently rinsed with deionized water to minimize experimental errors. After natural drying, uniform ginseng plant leaf samples were obtained using a punch, and the leaf area (S) and weight (M1) were measured. Subsequently, the leaf samples were immersed in the compound solution for 20 seconds, taken out, and allowed to drip off the excess liquid. After the dripping stopped, the samples were weighed (M2). This process was repeated five times, and the average value was calculated. The retention amount of the compound on the leaf samples before rinsing was calculated according to the following formula:

[0075]

[0076] The lower water contact angle and higher leaf liquid holding capacity indicate that the eugenol@nanozyme-sodium alginate hydrogel spray has good wettability and spreading properties, which can effectively reduce the loss of pesticides.

[0077] 2. Effects of the eugenol@nanozyme-sodium alginate hydrogel spray.

[0078] The peroxidase-like activity of the nanozyme was studied by the 3,3',5,5'-tetramethylbenzidine colorimetric method. The peroxidase-like catalytic performance of the nanozyme was measured in a 0.1 M acetic acid-sodium acetate buffer at pH 3.5. All reactions were carried out at 37 °C for 3 min. Among them, the nanozyme concentration was 10 μg / mL, and the total reaction system was 400 μL. By measuring the absorbance at 652 nm, the enzymatic kinetics of the nanozyme was studied, and the Lineweaver-Burk double reciprocal method was used to plot the graph. The results are as Figure 7 shown. The kinetic parameters K m and V max of TMB were 1.445 mM and 14.82 μΜ / min, respectively. The higher V max and lower K m emphasize its strong catalytic efficiency and applicability as an effective OXD mimic, indicating that the nanozyme has excellent peroxidase-like activity.

[0079] Disperse the eugenol@nanozyme-hydrogel sample containing 5 mg of eugenol@nanozyme in a release medium of 20 mL of PBES (PBS:ethanol = 3:1), and simulate in vitro release in a shaker at 25 °C at a rotation speed of 100 rpm. Extract 2 mL of the release medium at time intervals of 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 36 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h, and add the same volume of fresh release medium to ensure a constant release system. Centrifuge the obtained release medium, filter it through a disposable filter, and then measure the concentration of eugenol at a wavelength of 280 nm using a UV-visible spectrophotometer. Calculate the cumulative release amount of eugenol using the following formula:

[0080]

[0081] where EUcumulative release represents the cumulative release amount of eugenol, in %; V e is the volume of the release medium obtained at a given time interval, V e = 2 mL; V0 is the volume of the release solution, V0 = 20 mL; C n is the concentration of eugenol in the release medium at that time, in mg / L; m is the total mass of eugenol loaded in the nanozyme, in mg.

[0082] This invention studies the cumulative release behavior of the eugenol@nanozyme-sodium alginate hydrogel spray under the conditions of pH values of 3.5 and 7.4, and monitors it using a UV-visible spectrophotometer. The results are shown in Figure 8 . After 36 h of treatment, the cumulative release rate of the eugenol@nanozyme-sodium alginate hydrogel at pH 3.5 is 53.54%, and the cumulative release rate at pH 7.4 is 52.3%. As time increases, the release tends to slow down. The cumulative release rates at the 7th day are 76.8% and 73.9% respectively, indicating that the acidic condition does not hinder the release of the drug. After Botrytis cinerea infects the plant, it will release acidic compounds such as acetic acid. The acidic environment at the disease site not only does not affect the release of the active ingredients in the compound pesticide, but also enables the nanozyme to exhibit enzyme-like activity and play a synergistic antibacterial role.

[0083] 3. Biosafety detection of the eugenol@nanozyme-sodium alginate hydrogel spray

[0084] In the actual application process, the degradation of nanozymes will release various elements, so environmental safety is an important evaluation index. In this invention, the biosafety of nanozymes was evaluated using human epidermal cells. Human epidermal cells HaCat in the logarithmic growth phase were inoculated on 96-well plates and cultured at 37 °C for 24 h in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. The original culture medium was discarded, and the cells were cultured for another 24 h with a drug-containing culture medium containing nanoparticles at a concentration of 250 μg / mL. The cell viability was measured by the MTT method, and the results are shown in Figure 9 . It can be seen from Figure 9 that the nano-carrier has no obvious toxic effect on the growth of HaCat cells, indicating that the nanozyme nano-carrier has good biocompatibility and environmental safety.

[0085] Example 5

[0086] An in vitro antifungal activity test was carried out using Botrytis cinerea as the pathogen, and 6 groups were set up: a blank control group, an acetone solvent control group, a nanozyme experimental group, an eugenol experimental group, an eugenol@nanozyme experimental group, and an eugenol@nanozyme-sodium alginate hydrogel experimental group. The mycelial growth rate method was used to measure the antibacterial activity of the eugenol@nanozyme-sodium alginate hydrogel spray against Botrytis cinerea. Among them, the effective concentration of eugenol in the eugenol experimental group was 250 μg / mL, the effective concentration of eugenol in the eugenol@nanozyme experimental group was 200 μg / mL, and the effective concentration of nanozyme in the nanozyme experimental group was the same as that in the eugenol@nanozyme experimental group. The preparation methods of nanozyme and eugenol@nanozyme were the same as those in Example 1.

[0087] Under sterile conditions, Botrytis cinerea mycelial discs with a diameter of 6 mm were respectively inoculated in the center of potato dextrose agar culture dishes containing the above-mentioned concentration reagents. After culturing for 4 d at 25 °C in the dark, the colony diameter was measured by the cross method, and the relative inhibition rate against Botrytis cinerea was calculated. The test was repeated three times, and the results are as shown in Figure 10 .

[0088] During the growth process, Botrytis cinerea continuously secretes acetic acid, and the acidic condition is conducive to the nanozyme to exert its peroxidase-like activity. As shown in C of Figure 10 , eugenol itself, as a plant-derived pesticide, has a certain inhibitory effect on the growth of Botrytis cinerea, and its antibacterial effect reaches 46.06%. As shown in D of Figure 10 , the nanozyme will catalyze O2 to produce superoxide anions under acidic conditions, resulting in the inactivation of the fungal cell wall and membrane structure, and its antibacterial effect reaches 48.36%. As shown in Figure 10As shown by F in [reference], eugenol@nanozyme-sodium alginate hydrogel showed better inhibitory effect on Botrytis cinerea on PDA plates, with an inhibition rate of 100%, indicating the synergistic effect of nanozyme and eugenol in the nano-pesticide.

[0089] Example 6

[0090] Inspired by the in vitro anti-Botrytis cinerea activity of eugenol@nanozyme-sodium alginate hydrogel spray, the inhibitory effect of eugenol@nanozyme-sodium alginate hydrogel on mycelial growth was further evaluated using in vitro ginseng leaves. A blank control group, a nanozyme experimental group, an eugenol experimental group, and an eugenol@nanozyme-sodium alginate hydrogel experimental group were set up. An eugenol solution was prepared with 75% ethanol solution and diluted 20 times with deionized water to obtain 250 μg / mL. Then the SA-DA hydrogel precursor was dissolved in deionized water to obtain a precursor solution of 4 mg / mL. The eugenol@nanozyme was dispersed in the SA-DA hydrogel precursor solution to obtain a mixed suspension of nanozyme hydrogel precursor, in which the eugenol content was 200 μg / mL. It was used in combination with 2 wt% CaCl2 aqueous solution. First, the mixed suspension of nanozyme hydrogel precursor was sprayed, and then 2 wt% CaCl2 aqueous solution was sprayed. The concentration of nanozyme in the nanozyme experimental group was the same as that in the eugenol@nanozyme-sodium alginate hydrogel experimental group.

[0091] The leaves of 3-year-old ginseng plants were placed in 90 mm sterile petri dishes, and inoculated with a 5-day-old Botrytis cinerea mycelial disc with a diameter of 6 mm. The inoculated leaves were cultured in an incubator at a relative humidity of 85%, a constant temperature of 21 ± 1°C, a light period of 14 h, and a dark period of 10 h for 48 h. Then, 1 mL of the prepared fungicide solution was sprayed onto the surface of each leaf using a microsprayer. The control group used 1 mL of 75% ethanol solution diluted 20 times with deionized water, and each treatment was repeated 3 times. The leaves of each treatment group were placed back in the incubator for culture. Images of each leaf infected with Botrytis cinerea mycelial disc were taken with a camera, and the lesion area of each infected leaf was measured using Image J statistical analysis software (National Institutes of Health, USA). The inhibition rate of each treatment group was calculated according to the following formula:

[0092] Inhibition rate (%) = (A C - A T ) / (A C - A F ) × 100%;

[0093] where, A C is the lesion area of the control group, A T is the lesion area of each treatment group, and A F is the area of the mycelial disc.

[0094] The detection results of the anti-fungal activities of different pesticides on in vitro leaves are shown in Figure 11After 48 h of administration, the antifungal activity of the ginseng leaves in the eugenol@nanozyme-sodium alginate hydrogel treatment group was significant, and the damaged area was smaller than that of the eugenol group and the nanozyme group. The experimental results showed that the inhibition rates of the eugenol group and the nanozyme group were 38.36% and 39.52%, respectively, while the inhibition rate of the eugenol@nanozyme-sodium alginate hydrogel spray was 83.14%. This indicated that there was a synergistic effect between the nanozyme and eugenol in the nanopesticide, which was beneficial to reducing the dosage and increasing the efficiency.

[0095] The embodiments described above are only used to describe the preferred mode of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solution of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. A preparation method of eugenol@nanozyme for preventing and controlling gray mold of ginseng, characterized in that, It includes the following steps: Calcine ZIF-67 to obtain a nanozyme; Mix and react the nanozyme and eugenol in a solvent to prepare the eugenol@nanozyme.

2. The preparation method according to claim 1, characterized in that, The temperature of the calcination treatment is 600-800 °C, and the time is 3 h.

3. The preparation method according to claim 1, characterized in that, The mass ratio of the nanozyme to the eugenol is 1:4-6.

4. The preparation method according to claim 1, wherein The solvent is dimethyl sulfoxide.

5. An eugenol@nanozyme prepared by the preparation method according to any one of claims 1-4.

6. A preparation method of an eugenol@nanozyme-sodium alginate hydrogel spray for preventing and controlling gray mold of ginseng, which is characterized in that, It includes the following steps: Carry out a coupling reaction on sodium alginate and dopamine hydrochloride to prepare a DA-SA hydrogel precursor; Dissolve the DA-SA hydrogel precursor in water to obtain a precursor solution; Disperse the eugenol@nanozyme described in claim 5 in the precursor solution to obtain a nanozyme hydrogel precursor mixed suspension, which is the eugenol@nanozyme-sodium alginate hydrogel spray.

7. An eugenol@nanozyme-sodium alginate hydrogel spray prepared by the preparation method according to claim 6.

8. Use of an eugenol@nanozyme as described in claim 5 in the prevention and control of Botrytis cinerea of ginseng.

9. Use of an eugenol@nanozyme-sodium alginate hydrogel spray as described in claim 7 in the prevention and control of Botrytis cinerea of ginseng.

10. A method for preventing and controlling Botrytis cinerea of ginseng, characterized in that, It includes the step of sequentially spraying the eugenol@nanozyme-sodium alginate hydrogel spray described in claim 7 and an aqueous CaCl2 solution on the ginseng plants.

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