Pesticide nano-composite as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biochemistry, and provides a pesticide nano-composite and a preparation method and application thereof, the pesticide nano-composite comprises a dendritic polymer (SSP) and a drug molecule connected with the dendritic polymer through a hydrogen bond and / or Van der Waals force; the drug molecules are selected from at least one of benomyl, carbendazim and thiophanate-methyl; according to the drug compound, aggregation of drug molecules can be avoided, the drug effect of the drug molecules can be improved, the lasting period of the drug molecules can be prolonged, and the usage amount of the drug molecules is reduced; a solution containing the medicine compound is sprayed in the plant growth process and used for inhibiting growth of the aureocystis aureocystis and / or verticillium dahliae; the method is simple and convenient in process and wide in applicability.

Description

Technical Field

[0001] The present invention relates to the field of biochemistry technology, and particularly to a pesticide nano - composite, a preparation method thereof and an application thereof. Background Art

[0002] The wide application of chemical pesticides in the prevention and control of agricultural pests and diseases is of great significance. However, their high dosage and low utilization rate lead to environmental pollution and economic losses. Existing research shows that nanotechnology can significantly improve the performance of pesticides by enhancing their dispersibility and stability. For example, dendritic nano - carriers can be ideal carriers for pesticides due to their high specific surface area and functionalizable characteristics. Nanoparticles refer to ultrafine particles with a particle size between 1 - 100 nm. Due to their unique three - dimensional scale, they have characteristics such as small size, large specific surface area, strong modifiability, good aqueous solution dispersibility, strong adhesion, and photocatalytic degradation. Using nano - carriers is expected to improve the bactericidal performance of pesticides and enhance the control effect of diseases. However, there are few existing nano - carriers suitable for the prevention and control of forest pests and diseases, and the control effect on diseases needs to be further improved. Summary of the Invention

[0003] In order to solve the above problems to a certain extent, the present invention provides a pesticide nano - composite, a preparation method thereof and an application thereof.

[0004] Specifically, in the first aspect, the present invention provides a drug composite, which includes a dendritic polymer (SSP) and drug molecules connected to the dendritic polymer by hydrogen bonds and / or van der Waals forces; the drug molecules are selected from at least one of benomyl, carbendazim, and thiophanate - methyl; the dendritic polymer has the structure shown in Formula 1 below: ; Wherein, n is a positive integer, preferably an integer from 1 to 100. The specific value of n depends on the degree of polymerization. During the reaction process, by controlling the reaction conditions, the degree of polymerization is controlled to achieve the control of the n value.

[0005] The drug composite provided by the present invention is a composite of drug molecules and a nano - carrier. The nano - carrier and drug molecules are combined together through hydrogen bonds and van der Waals forces, overcoming the problems that existing pesticides have low solubility in water and are prone to agglomeration, which is not conducive to plant absorption; at the same time, the dendritic structure of the nano - carrier is beneficial to the loading of pesticides, forming a stable composite, and the increased dendritic structure can effectively improve the loading capacity of pesticides; at the same time, after the nano - carrier is functionalized with amino functional groups (loading drug molecules), it can effectively reduce the particle size of the loaded substances (such as drug molecules), promote the penetration of pesticides through the epidermal barrier of forest trees, increase the absorption amount of forest trees to pesticides, and thus play an efficient bactericidal role.

[0006] Using the dendritic polymer provided by the present invention as a carrier can increase the loading amount, and it is inexpensive and easy to synthesize, making it more suitable for applications in forestry production.

[0007] The dendritic polymer of the present invention can also be used as a carrier for substances such as nucleic acid molecules.

[0008] According to the drug complex provided by the present invention, the loading amount of the drug molecule is more than 6%.

[0009] According to the drug complex provided by the present invention, the average particle size of the drug complex is 40 nm or less. The small average particle size of the drug complex of the present invention is more conducive to penetrating the epidermal barrier of forest trees and entering the interior of forest trees, thereby playing an efficient antibacterial role.

[0010] Preferably, when the drug molecule is selected from benomyl, the particle size of the drug complex is 15 nm or less. Specifically, the average particle size of the drug complex can be 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, etc.

[0011] Preferably, when the drug molecule is selected from carbendazim, the particle size of the drug complex is 35 nm or less. Specifically, the average particle size of the drug complex can be 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 25 mm, 35 mm, etc.

[0012] According to the drug complex provided by the present invention, the PDI of the dendritic polymer is 1 to 1.2; the molecular weight of the dendritic polymer is 3.0×10³ to 3.4×10³ g / mol.

[0013] According to the drug complex provided by the present invention, the preparation method of the dendritic polymer includes: dropping a THF solution of 2-bromo-2-methylpropionyl bromide into a THF solution of ditrimethylolpropane with a pH of 6.5 to 7.5 at a temperature of 0 °C to 5 °C, and obtaining an initiator after sufficient reaction; mixing the initiator, a halide of Cu + , N,N-dimethylaminoethyl methacrylate and THF, and then adding N,N,N',N',N''-pentamethyldiethylenetriamine, and carrying out an atom transfer radical polymerization reaction under an inert atmosphere to obtain the dendritic polymer.

[0014] According to the drug complex provided by the present invention, the concentration of the anhydrous THF solution of 2-bromo-2-methylpropionyl bromide is 1.5 to 2.0 M.

[0015] According to the drug complex provided by the present invention, the concentration of the anhydrous THF solution of ditrimethylolpropane is 0.04 to 0.06 M.

[0016] For the pharmaceutical complex provided by the present invention, the molar ratio of the 2-bromo-2-methylpropanoyl bromide to the ditrimethylolpropane is 20 to 30:1.

[0017] For the pharmaceutical complex provided by the present invention, the molar ratio of the initiator to the halide of Cu + is 0.05 to 0.06:0.3 to 0.4.

[0018] For the pharmaceutical complex provided by the present invention, the molar ratio of the initiator to the N,N-dimethylaminoethyl methacrylate is 0.05 to 0.06:11 to 12.

[0019] For the pharmaceutical complex provided by the present invention, the Cu + halide and the N,N,N',N',N''-pentamethyldiethylenetriamine have a molar ratio of 0.3 to 0.4:0.6 to 0.7. Among them, the Cu + halide can be CuBr, CuCl, CuI, etc.

[0020] For the pharmaceutical complex provided by the present invention, the molar volume ratio of the initiator to the THF is 0.05 to 0.06 mmol:15 to 20 mL.

[0021] For the method for preparing the dendritic polymer of the pharmaceutical complex provided by the present invention, it includes the following steps: (1) Under an inert atmosphere, dissolve ditrimethylolpropane in anhydrous THF to obtain an anhydrous THF solution of ditrimethylolpropane; add triethylamine dropwise at room temperature until the pH reaches 7; at 0°C to 5°C, add an anhydrous THF solution of 2-bromo-2-methylpropanoyl bromide at a rate of 20 to 60 drops per minute while maintaining stirring; heat to room temperature and stir for 20 to 40 h; quench the reaction and recrystallize to obtain the initiator (2-(2-bromoethoxy)anisole Dh-Br).

[0022] Among them, the addition rate of the anhydrous THF solution of 2-bromo-2-methylpropanoyl bromide can be 20 drops, 30 drops, 40 drops, 50 drops, 60 drops, etc. per minute; it can be carried out at a temperature of 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, and its concentration can be 1.5 mo / L, 1.6 mol / L, 1.7 mol / L, 1.8 mol / L, 1.9 mol / L, 2.0 mol / L, etc.

[0023] Preferably, the addition of the anhydrous THF solution of 2-bromo-2-methylpropanoyl bromide is completed within 25 to 35 minutes.

[0024] Among them, heating to room temperature is completed within 30 to 40 minutes, such as 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, etc. The reaction time can be any value within 20 - 40 hours, such as 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, etc.

[0025] Among them, recrystallization can be carried out in diethyl ether.

[0026] (2) Using atom transfer radical polymerization method, N,N-dimethylaminoethyl methacrylate is used as the monomer, and the initiator is added, and the dendritic polymer is synthesized through a polymerization reaction.

[0027] The process of step (2) is as follows: Add the initiator (Dh-Br), Cu + halide and N,N-dimethylaminoethyl methacrylate (DMAEMA) into THF. After dissolving and mixing evenly, add N,N,N',N',N''-pentamethyldiethylenetriamine (PMDETA), and carry out three freeze-thaw cycles to remove oxygen; Stir at 50 - 70 °C for 8 - 12 hours to carry out the polymerization reaction; Terminate the reaction and remove THF, dialyze, and freeze-dry to obtain the dendritic polymer.

[0028] Preferably, in steps (1) and (2), the termination reaction includes adding methanol and / or exposing to air. Among them, the specific operation of quenching the reaction is: Stop heating, add a small amount of methanol to the flask, connect to air, and wait for 0.5 - 1 h for the temperature to drop to room temperature; The amount of methanol is generally consistent with the molar amount of the initiator.

[0029] Preferably, in step (2), the temperature of the polymerization reaction can be 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, etc.; The stirring time can be 8 h, 9 h, 10 h, 11 h, 12 h, etc.

[0030] In the second aspect, the present invention also provides a preparation method of the drug complex as described above, including: Complexing the dendritic polymer with drug molecules in a solution system.

[0031] According to the preparation method of the drug complex provided by the present invention, the mass ratio of the dendritic polymer to the drug molecule is 1:10 to 10:1.

[0032] According to the preparation method of the drug complex provided by the present invention, the dendritic polymer and the drug molecule are dissolved in water and complexed at 15 - 35 °C.

[0033] The present invention also provides the application of the said drug complex in reducing the amount of pesticides used for pest control. In particular, it can achieve the effect of reducing the amount of pesticides used while increasing the efficiency in controlling forest diseases, providing a new method for the prevention and control of forest diseases. Specifically, in the third aspect, the present invention also provides the application of the drug complex as described above, including: spraying a solution containing the drug complex during the growth process of plants to inhibit the growth of Cytospora chrysosperma and / or Verticillium dahliae; the content of the drug molecules in the solution of the drug complex is above 0.01‰.

[0034] Preferably, the effective amount of the drug complex is 600 - 650 grams per hectare. For example, in some embodiments, it is 611.7 grams per hectare, which is 22.6% less than 790.3 grams per hectare when applying pesticides alone.

[0035] As an example, the application of the drug complex includes: spraying a solution containing the drug complex on the bark of poplar trees during the growth process of poplar trees.

[0036] It has been verified that when the drug molecule is benomyl, the improvement in loading amount, bactericidal effect, duration, and reduction in dosage is more obvious.

[0037] The pesticide nano - complex provided by the present invention, its preparation method and application, are such that a dendritic polymer with a specific structure and drug molecules are combined through hydrogen bonding and van der Waals force interactions. The drug molecule loading amount is high, and the formed drug complex can avoid the aggregation of drug molecules. Especially for benomyl, its particle size is reduced from 864 nm to below 15 nm, which is crucial for enhancing the effect and duration of benomyl and reducing its usage amount; under specific conditions, this complex significantly improves the growth inhibition rate of pathogenic bacteria (for example, in some embodiments, benomyl is increased by 18.8% and carbendazim is increased by 22.6%), and at the same time, the amount of pesticides used can be reduced by 22.6%.

[0038] The dendritic polymer provided by the present invention has good dispersibility and stability in aqueous solution, which is more conducive to the toxicity and persistence of pesticides.

[0039] The preparation method of the drug complex provided by the present invention has a simple operation process during the configuration. The process is simple and fast. Just mix well according to the recommended mass ratio and let it stand for 15 minutes, then a stable complex can be formed for conventional spraying operations. It has a wide applicability, is easy to promote and popularize, and is suitable for the green prevention and control of various chemical pesticides.

[0040] The present invention has a wide range of applications. For different types of pesticides, it can improve their toxicity to varying degrees and extend their persistence period. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0042] Figure 1 1H NMR spectrum of the initiator in the preparation process of SSP provided in Preparation Example 1 of the present invention.

[0043] Figure 2 1H NMR spectrum of SSP provided in Preparation Example 1 of the present invention.

[0044] Figure 3 GPC chart of SSP provided in Preparation Example 1 of the present invention.

[0045] Figure 4 Isothermal titration calorimetry detection results of benomyl-nanocarrier complex provided in Test Example 2 of the present invention.

[0046] Figure 5 Isothermal titration calorimetry detection results of carbendazim-nanocarrier complex provided in Test Example 2 of the present invention.

[0047] Figure 6 Electron microscopy detection result diagrams of benomyl and benomyl-nanocarrier complex in Test Example 3 of the present invention; wherein, a is the electron microscopy photo of benomyl, b is the electron microscopy photo of benomyl-nanocarrier complex, c is the particle size distribution diagram of benomyl, and d is the particle size distribution diagram of benomyl-nanocarrier complex.

[0048] Figure 7 Electron microscopy detection result diagrams of carbendazim and carbendazim-nanocarrier complex in Test Example 3 of the present invention; wherein, a is the electron microscopy photo of carbendazim, b is the electron microscopy photo of carbendazim-nanocarrier complex, c is the particle size distribution diagram of carbendazim, and d is the particle size distribution diagram of carbendazim-nanocarrier complex.

[0049] Figure 8 Test result diagrams of the virulence detection of different samples on Cytospora chrysosperma in Test Example 5 of the present invention. Among them, a is the growth schematic diagram of Cytospora chrysosperma on the PDA plate, b is the growth diameter diagram of Cytospora chrysosperma under different treatments, and c is the statistical chart of the growth inhibition rate of different treatments on Cytospora chrysosperma.

[0050] Figure 9 Diagrams of the disease incidence of poplar branches under different treatments in Test Example 5 of the present invention.

[0051] Figure 10Schematic diagram for detecting the control effects of different treatments by the spraying method in Test Example 5 of the present invention. Among them, a is a physical picture of poplar branches after different treatments, b and d are diagrams of the growth diameter of branch disease spots, and c and e are diagrams of the inhibition rate of branches.

[0052] Figure 11 Test result diagram for detecting the virulence of Verticillium dahliae by different treatments in Test Example 6 of the present invention. Among them, a is a schematic diagram of the growth of Verticillium dahliae on a PDA plate, b is a diagram of the growth diameter of Verticillium dahliae under different treatments, and c is the statistical chart of the growth inhibition rate of different treatments on Verticillium dahliae.

[0053] Figure 12 Test result diagram for Test Example 7 of the present invention. Among them, a is a physical picture of poplar branches after different treatments, b and d are diagrams of the growth diameter of branch disease spots, and c and e are diagrams of the inhibition rate of branches. Detailed implementation manners

[0054] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below with reference to the accompanying drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0055] For those not specifying specific technologies or conditions in the embodiments, they shall be carried out according to the technologies or conditions described in the literature in the field or according to the product specifications. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0056] The present invention will be described below with reference to specific embodiments. It should be noted that these embodiments are only descriptive and do not limit the present invention in any way.

[0057] Preparation Example 1 This preparation example provides a preparation method for an amino-functionalized dendritic polymer, and the specific steps are as follows: (1) Under nitrogen, pentaerythritol bis(pentaerythritol) (1 mmol) was dissolved in anhydrous THF (20 mL) in a 250 mL round-bottom flask. Triethylamine (9.34 mmol) was added dropwise at room temperature to neutralize the acidic by-products formed during the reaction. At 0 °C, a solution of freshly prepared 2-bromo-2-methylpropanoyl bromide (25.51 mmol) in anhydrous THF (15 mL) was slowly added to the mixture at a dropping rate of 40 drops / min over 30 minutes while maintaining constant stirring. The reaction mixture was then gradually warmed to room temperature and stirred overnight to ensure complete reaction. After completion, the reaction was quenched by adding methanol to neutralize the excess bromide. The crude product was purified by recrystallization from cold diethyl ether to obtain an initiator in the form of a white crystalline powder, denoted as Dh-Br. Its 1H NMR spectrum is as shown in Figure 1 follows: 1H NMR (400 MHz, CDCl3, Bruker 400) δ 4.33 (s, 8H), 1.94 (s, 24H).

[0058] (2) A six-armed star polymer was synthesized by atom transfer radical polymerization (ATRP). The specific procedure was as follows: In a dry Schlenk flask, Dh-Br (0.055 mmol), CuBr (0.33 mmol), DMAEMA (11.55 mmol) and THF (16 mL) were added, and then PMDETA (0.66 mmol) was introduced as a ligand to stabilize the copper catalyst. The reaction flask was subjected to three freeze-pump-thaw cycles to remove oxygen, ensuring an inert atmosphere crucial for ATRP. The polymerization reaction was carried out at 60 °C with constant stirring for 10 hours. To terminate the polymerization, the reaction was cooled to room temperature and exposed to air. The solvent (THF) was removed under reduced pressure using a rotary evaporator, and the crude product was purified by dialysis in deionized water for three days. The water was changed regularly until the dialysis solution was clear. After freeze-drying, a purified polymer, denoted as SSP, was obtained as a white powder. The structure of SSP was confirmed by 1 1H NMR (400 MHz, D 2 2O, Bruker 400).

[0059] The 1H NMR results of the initiator and polymer provided in this preparation example are as shown in Figure 1 and Figure 2 respectively. It can be seen from Figure 1 that a peak was shown at δ ≈ 4.3 ppm, corresponding to the hydrogen signal of the methoxy group (CH 2 2O) in the pentaerythritol core; an obvious peak was observed at δ ≈ 1.9 - 2.0 ppm, attributed to the methyl hydrogens in the 2-bromo-2-methylpropanoic acid groups on each arm. The integral ratio of each peak was consistent with the number of hydrogens calculated theoretically in the molecular structure, verifying the successful synthesis of the target compound.

[0060] In addition, no obvious impurity peaks were observed in the spectrum, indicating that the sample had a high purity.

[0061] These results indicated that the six-armed star initiator had been successfully synthesized with a definite structure, providing a reliable basis for subsequent applications.

[0062] From Figure 2 it can be seen that the peaks located at δ≈2.5 - 3.0 ppm corresponded to the aminomethyl (-CH 2 N(CH 3 ) 2 ) in the side chain of polyDMAEMA and the methyl groups (-CH 2 C(O)O-) near the ester group. Due to the influence of the chemical environment of the adjacent amino and carbonyl groups, these hydrogen atoms showed a chemical shielding effect, resulting in their chemical shifts being distributed in the higher field region. The peak at δ≈1.9 ppm was attributed to the branched-chain methyl of 2-bromo-2-methyl propionate in the six-armed star structure. This peak was broad but had a significant signal, reflecting the combined influence of the bromine atom and the carbonyl group on the chemical environment.

[0063] Overall, the positions and integral ratios of the chemical shifts were highly consistent with the theoretical structure of the target compound, further verifying the successful synthesis of the target compound and indicating that the sample had a high purity and structural integrity.

[0064] Figure 3 The GPC results showed that the main peak molecular weight of the polymer met the expectation of forming a medium molecular weight polymer by ATRP reaction. In addition, the molecular weight distribution curve presented typical polymer characteristics, and no obvious low molecular weight monomer or oligomer residues were observed, indicating that the monomers had fully participated in the polymerization reaction. At the same time, the cumulative distribution curve increased smoothly, indicating that the molecular weight distribution of the obtained polymer was reasonable and there were no abnormal high molecular weight components. In addition, this polymerization reaction adopted the ATRP mechanism, starting from the initiator and proceeding with controlled radical polymerization under the CuBr / PMDETA catalytic system. The molecular weight distribution (PDI = 1.10) shown by the GPC data further verified the controllability of this polymerization system, indicating that no significant chain termination or branching occurred during the reaction process.

[0065] Combined with the ¹H NMR data (the characteristic signals of -OCH 2 - and -N(CH 3 ) 2 ) and the GPC results (molecular weight growth and reasonable molecular weight distribution), it could be confirmed that the target polymer had been successfully synthesized, and the polymerization reaction had a high conversion rate and good controllability. Therefore, it could be determined that the product prepared in this preparation example had the structure shown in Formula 1 below: .

[0066] In Formula 1, n is a positive integer.

[0067] Test Example 1 Loading Rate Test Object: The carrier aqueous solution formed by the product obtained in Preparation Example 1.

[0068] The carrier aqueous solution formed by the polymer in Example 1 of CN110432263B.

[0069] Test Method: A series of benomyl and carbendazim standard solutions with concentrations of 0, 2.5 μg / mL, 5 μg / mL, 7.5 μg / mL, 10 μg / mL, 12.5 μg / mL, and 15 μg / mL were prepared using chloroform. The ultraviolet absorbance of these solutions was measured at the characteristic wavelength using a UV-Vis spectrophotometer (UV-2600, Shimadzu, Japan). A calibration curve was constructed for each pesticide, correlating the absorbance with the concentration.

[0070] For the loading capacity experiment, benomyl and carbendazim were dissolved in chloroform and incubated with the above-mentioned carrier aqueous solution at a volume ratio of 1:1. After incubating at room temperature for 15 minutes, unbound pesticides were removed using a regenerated cellulose dialysis bag (cut-off molecular weight: 3500 Da). Then the dialysis solution was lyophilized and redissolved in a solvent for quantification by UV-Vis spectrophotometry.

[0071] Test Results: See Table 1.

[0072] Table 1

[0073] As can be seen from Table 1, compared with the nano-carrier used in CN10432263B, the method of the present invention can obtain a higher loading rate, and the loading effect for benomyl is better.

[0074] Test Example 2 Isothermal Titration Calorimetry Detection Sample Preparation 1: Carbendazim was dissolved in acetonitrile to obtain a carbendazim solution with a mass concentration of 0.04 μM; SSP was dissolved in water to obtain an SSP solution with a mass concentration of 0.25 mM; at 25 °C, 50 μL of the SSP solution (0.25 mM) was titrated into 300 μL of the carbendazim solution. During each injection, the ΔG value was calculated using Origin 7 software and the following formula.

[0075] Sample preparation 2: Benomyl was dissolved in acetonitrile to obtain a benomyl solution with a mass concentration of 0.04 μM; SSP was dissolved in water to obtain an SSP solution with a mass concentration of 0.25 mM; at 25 °C, 50 μL of the SSP solution (0.25 mM) was titrated into 300 μL of the benomyl solution. During each injection, the ΔG value was calculated using Origin 7 software and the following formula.

[0076] Test: Isothermal titration calorimetry was used to detect the binding process between the above-mentioned drug molecules and the carrier, and the test results are as Figures 4 - 5 shown.

[0077] It can be seen from Figure 4 that the interaction between benomyl and SSP was studied by isothermal titration calorimetry (ITC). The high binding constant (Ka of benomyl = 1.80×10 5 M) and the negative Gibbs free energy change (ΔG of benomyl = -27.08 kJ / mol) indicate spontaneous and strong binding. The negative enthalpy (ΔH) and negative entropy (ΔS) values confirm that hydrogen bonding and van der Waals forces are the dominant mechanisms driving the self-assembly of the complex.

[0078] It can be seen from Figure 5 that the interaction between carbendazim and SSP was studied by isothermal titration calorimetry (ITC). The high binding constant (Ka of carbendazim = 5.08×10 5 M) and the negative Gibbs free energy change (ΔG of carbendazim = -24.40 kJ / mol) indicate spontaneous and strong binding. The negative enthalpy (ΔH) and negative entropy (ΔS) values confirm that hydrogen bonding and van der Waals forces are the dominant mechanisms driving the self-assembly of the complex.

[0079] Therefore, the complex formed by benomyl and SSP has the structure shown in Formula 2 below:

[0080] The complex formed by carbendazim and SSP has the structure shown in Formula 3 below: .

[0081] Test Example 3 Transmission Electron Microscopy Test Sample preparation 1: Benomyl was dissolved in ethanol to obtain a benomyl solution with a mass concentration of 0.3 g / L; SSP was dissolved in ethanol to obtain an SSP solution with a mass concentration of 0.3 g / L; according to a mass ratio of benomyl to SSP of 1:1, the benomyl solution and the SSP solution were mixed, stirred evenly, and incubated at room temperature for 15 minutes to form a solution containing the complex.

[0082] Sample preparation 2: It is basically the same as Sample preparation 1, except that benomyl is replaced with carbendazim in equal mass.

[0083] Test: The transmission electron microscopy results of benomyl and the complex are shown in a and b of Figure 6 , and the particle size distributions are shown in c and d. As can be seen from Figure 6 c of Figure 6 , the average particle size of benomyl is 864.36 nm; as can be seen from

[0084] d of Figure 7 , the average particle size of the complex is 14.76 nm. Figure 7 Figure 7

[0085]

[0086] It shows that the method provided by the present invention can effectively reduce the particle size of drug molecules.

[0086] Test Example 4 Stability Since the polymer can maintain good stability at extreme temperatures, is not prone to swelling and deformation, is beneficial to maintaining the stability of the pesticide nanocarrier complex, extending the service life of the complex and using it in more complex environmental conditions, therefore, the stability of SSP is evaluated by examining the particle size change of SSP under different temperature conditions.

[0087] Sample preparation: Dissolve SSP in water.

[0088] Test: Use a particle size analyzer (Anton Paar Litesizer 500, Austria) to measure the particle size and polydispersity index (PDI). Each treatment includes 3 independent samples.

[0089] The test results are shown in Table 2.

[0090] Table 2

[0091] Use analysis of variance (ANOVA) to analyze the data at a significance level of P = 0.05.

[0092] As can be seen from Table 2, at different temperatures, the dispersity of SSP is within a reasonable distribution range. At this time, whether in extreme environmental temperatures or at room temperature, the particle size of the polymer remains between 10 and 15 nm, without significant fluctuations. These results indicate that the polymer can also maintain stability at extreme temperatures and has a wide application temperature range.

[0093] Test Example 5 Virulence Detection against Cytospora chrysosperma Test object: The SSP (also known as the nanocarrier) obtained in Preparation Example 1; Benomyl; Carbendazim; Complex solution A formed by benomyl and the carrier: According to the mass ratio of benomyl to SSP being 1:1, benomyl and SSP were dissolved in water, mixed, stirred evenly, and incubated at room temperature for 15 minutes to form solution A containing the complex.

[0094] Complex solution formed by carbendazim and the carrier: It is basically the same as solution A, except that: benomyl was replaced with carbendazim in equal mass to obtain solution B.

[0095] Test method: (1) Benomyl and carbendazim were respectively diluted with sterile water at gradient concentrations to obtain their median inhibitory concentrations against Cytospora chrysosperma. Specifically, the median inhibitory concentration of benomyl was 3.8 mg / L, and the median inhibitory concentration of carbendazim was 2.6 mg / L.

[0096] (2) Sample preparation: Benomyl solution diluted with sterile water to the median inhibitory concentration.

[0097] Carbendazim solution diluted with sterile water to the median inhibitory concentration.

[0098] Solution A was diluted with sterile water to solution A1 at the median inhibitory concentration of benomyl.

[0099] Solution B was diluted with sterile water to solution B1 at the median inhibitory concentration of carbendazim.

[0100] SSP was mixed with sterile water, and the mass concentration of SSP was 1 mg / mL.

[0101] (3) The test samples were added to PDA plates inoculated with Cytospora chrysosperma strains. Three plates were inoculated at a time, and it was repeated 5 times. Observe and record the growth of Cytospora chrysosperma, and record the change in its growth diameter. Test results: As shown in a - c of Figure 8 .

[0102] Combined with Figure 8 a - c in it, it can be seen that compared with the single application of carbendazim and benomyl, the growth inhibition rates of the complex against Cytospora chrysosperma were increased by 15.7% and 18.8% respectively, indicating that the toxicity of the present invention is significantly improved compared with general pesticides.

[0103] Furthermore, as shown in Figure 9As shown, Cytospora chrysosperma was inoculated on healthy poplar branches. After 48 hours of colonization, solution A2 (solution A diluted with sterile water to a concentration of 0.05‰ benomyl) was sprayed. 25 branches were inoculated each time, and the experiment was repeated 3 times. The medicament was sprayed 1 day, 3 days, and 5 days after the colonization of Cytospora chrysosperma. On the 7th day, the disease incidence of poplar branches was observed and recorded, and the lesion size was recorded. The data were from three repeated experiments, and the statistical significance analysis was performed using SPSS software, with the significance level set at p < 0.05. At the same time, the following solutions were used to replace solution A2 respectively to repeat this step: Benomyl solution diluted with sterile water to a concentration of 0.05‰; Carbendazim solution diluted with sterile water to a concentration of 0.05‰; Solution B2: solution B diluted with sterile water to a concentration of 0.05‰ carbendazim; SSP solution: SSP and sterile water were mixed, and the mass concentration of SSP was 1 mg / mL.

[0104] Sterile water.

[0105] Test results: As shown in a - e of Figure 10 , where CK: blank control, WT: negative control (wild - type group).

[0106] Combined with Figure 10 It can be seen that after benomyl and carbendazim are combined with the nanocarrier, the growth inhibition rate and control effect on Cytospora chrysosperma are both improved. Among them, the control effect of benomyl is improved by 16.4%, and the control effect of carbendazim is improved by 22.6%. It shows that spraying the complex medicament by the method described in the present invention can effectively improve the toxicity of pesticides.

[0107] Test Example 6 Toxicity Detection for Verticillium dahliae Test method: (1) Benomyl was diluted with sterile water at gradient concentrations, and the median inhibitory concentration against Verticillium dahliae was obtained as 4 μg / ml.

[0108] (2) Sample preparation: 4 μg / ml benomyl: Benomyl was diluted with sterile water to the median inhibitory concentration.

[0109] 4 μg / ml benomyl - nanocarrier complex: Solution A was diluted with sterile water to the median inhibitory concentration of benomyl to obtain solution A3.

[0110] 4 μg / ml benomyl / SPc: The preparation process was basically the same as that of 4 μg / ml benomyl - nanocarrier complex, except that: SSP was replaced with SPc in equal mass.

[0111] Nanocarrier solution: Mix SSP and sterile water, with the mass concentration of SSP being 1 mg / mL.

[0112] SPc solution: The preparation process is basically the same as that of the nanocarrier solution, except that SSP is replaced with SPc of equal mass.

[0113] Sterile water (3) Add the sample to be tested to the PDA plate and inoculate the Verticillium dahliae strain. Inoculate 3 plates at a time and repeat 5 times.

[0114] (4) Observe and record the growth of Verticillium dahliae after inoculating the plate, and record the change in its growth diameter. Calculate the growth inhibition rate of the liquid medicine on Verticillium dahliae.

[0115] Test results: As Figure 11 shown. It can be seen that the growth inhibition rate of the complex of the present invention on Verticillium dahliae has increased by 28.8%, indicating that the toxicity of the present invention is significantly improved compared with general pesticides.

[0116] Combined with the toxicity test results and the foregoing analysis of the loading capacity and reduction of the drug molecule particle size, it can be known that using SSP provided in Preparation Example 1 of the present invention as a drug molecule carrier can effectively increase the absorption amount of the drug molecule by trees, thereby improving its sterilization performance.

[0117] Test Example 7 Dosage of the drug molecule-carrier complex Test method: Inoculate Cytospora chrysosperma on healthy poplar branches. After 48 hours of colonization, spray Solution A4 (dilute Solution A with sterile water to obtain Solution A4 with the concentration of benomyl being 0.01‰). Inoculate 25 branches at a time and repeat 3 times. Spray the liquid medicine 1 day, 3 days, and 5 days after the colonization of Cytospora chrysosperma, and observe and record the disease condition of the poplar branches on the 7th day, and record the size of the lesions.

[0118] Repeat this step by replacing Solution A4 with the following solutions respectively: Dilute benomyl with sterile water to a benomyl solution with a concentration of 0.01‰; Dilute carbendazim with sterile water to a carbendazim solution with a concentration of 0.01‰; Dilute Solution B with sterile water to obtain Solution B4 with the concentration of carbendazim being 0.01‰; SSP solution: Mix SSP and sterile water, with the mass concentration of SSP being 1 mg / mL.

[0119] Sterile water.

[0120] Test results: As Figure 12 shown. Among them, CK: blank control, WT: negative control.

[0121] FromFigure 12 It can be seen that after the colonization of Cytospora chrysosperma, the application of low concentrations of carbendazim and benomyl alone can no longer achieve obvious control effects on poplar branches. The growth inhibition rate and control effect of Cytospora chrysosperma have both increased after the combination of benomyl and carbendazim with the nanocarrier. Among them, the control effect of benomyl has increased by 58.6%, and the control effect of carbendazim has increased by 54.7%. This shows that spraying the solution of the composite by the method described in the present invention can effectively improve the toxicity of pesticides at low concentrations and can effectively reduce the amount of pesticides used.

[0122] It should be understood that the technical solutions obtained by proportionally enlarging or reducing the amounts of the reagents or raw materials used in the above embodiments are substantially the same as those of the above embodiments.

[0123] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A drug complex, characterized in that The invention comprises a dendritic polymer and a drug molecule connected to the dendritic polymer by hydrogen bonds and / or van der Waals forces; the drug molecule is selected from at least one of benomyl, carbendazim and methylthiophanate; the dendritic polymer has a structure shown in the following formula 1: ； Wherein, n is a positive integer.

2. The drug complex according to claim 1, characterized in that: The loading amount of the drug molecules is more than 6%.

3. The drug complex according to claim 1, characterized in that: The average particle size of the drug complex is less than 40 nm.

4. The drug complex according to claim 1, characterized in that: The PDI of the dendritic polymer is 1-1.2; the molecular weight of the dendritic polymer is 3.0×10³-3.4×10³ g / mol.

5. The drug complex according to any one of claims 1 to 4, characterized in that The preparation method of the dendritic polymer comprises: At a temperature of 0°C to 5°C, a THF solution of 2-bromo-2-methylpropionyl bromide is added dropwise to a THF solution of dipentaerythritol at a pH of 6.5 to 7.5, and an initiator is obtained after sufficient reaction; The initiator, Cu + The halide, N,N-dimethylaminoethyl methacrylate and THF are mixed, and N,N,N',N',N''-pentamethyldiethylenetriamine is added to carry out an atom transfer radical polymerization reaction under an inert atmosphere to obtain the dendritic polymer.

6. The drug complex according to claim 5, characterized in that: The concentration of the THF solution of 2-bromo-2-methylpropionyl bromide is 1.5-2.0 M; The concentration of the dipentaerythritol THF solution is 0.04-0.06M; The molar ratio of the 2-bromo-2-methylpropionyl bromide to the dipentaerythritol is 20-30:1; The initiator and the Cu + The molar ratio of the halide is 0.05~0.06:0.3~0.4; The molar ratio of the initiator to the N,N-dimethylaminoethyl methacrylate is 0.05-0.06:11-12; The Cu + The molar ratio of the halide to the N,N,N',N',N''-pentamethyldiethylenetriamine is 0.3-0.4:0.6-0.7; The molar volume ratio of the initiator to the THF is 0.05-0.06 mmol:15-20 mL.

7. The method for preparing the drug complex according to any one of claims 1 to 6, characterized in that: include: The dendrimers are complexed with drug molecules in a solution system.

8. The method for preparing the drug complex according to claim 7, characterized in that: The mass ratio of the dendrimer to the drug molecule is 1:10 to 10:

1.

9. The method for preparing the drug complex according to claim 7, characterized in that: The dendrimer and drug molecules were dissolved in water and complexed at 15-35 °C.

10. Use of the drug complex according to any one of claims 1 to 6 or the drug complex prepared by the preparation method according to any one of claims 7 to 9, characterized in that: include: The solution containing the drug complex is sprayed during the growth of plants to inhibit the growth of Cystosporum chrysanthemi and / or Verticillium dahliae; the content of drug molecules in the solution of the drug complex is above 0.01‰.

Images