Tebuconazole-containing coordination polymerization preparation method and sustained-release antibacterial application

Abstract

The invention relates to a tebuconazole-containing coordination polymerization preparation method and a sustained-release antibacterial application. According to the coordination polymer, zinc is used as a central metal, ligands are tebuconazole and HCOO-R-COOH, R is selected from at least one of a benzene ring, a heterocyclic ring and an aliphatic group, and a double-ligand type coordination polymer is formed. The preparation method of the coordination polymer is any one of the preparation methods of the coordination polymer. Zinc salt, the HCOO-R-COOH and the tebuconazole are sequentially added into a DMF aqueous solution, stirring is conducted for 0.1-72 h under the oil bath condition of 0-200 DEG C, and the coordination polymer is obtained. The zinc salt comprises at least one of zinc chloride, zinc sulfate and zinc nitrate. The coordination polymer provided by the invention has ultrahigh bactericide load, excellent bactericidal effect and better anti-ultraviolet stability, and shows dual-response release in the aspects of pH value and temperature.

Description

Technical Field

[0001] The invention belongs to the technical field of metal coordination polymers, and particularly relates to a preparation method of a coordination polymer containing tebuconazole and its sustained-release and antibacterial application. Background Art

[0002] Fungicides play an indispensable role in controlling crop pests and diseases in modern agriculture. However, due to a variety of factors such as their physicochemical properties (such as water solubility and chemical stability), application techniques (such as spraying and fumigation) and environmental factors (such as wind speed and temperature), up to 90% of fungicides are degraded or volatilized into the environment before reaching the target location. Fungicides diffused into the environment may also eventually enter the bodies of farmers and farm workers, affecting their health. In addition, fungicides successfully sprayed on plants are further wasted due to rapid hydrolysis, photolysis or microbial metabolism. Fungicide losses due to degradation may exacerbate environmental problems. All of the above fungicide residues and metabolites may increase food safety risks.

[0003] Compared to typical one-time spraying of fungicides, fungicides with controlled-release (CR) capabilities can significantly prolong their effectiveness and reduce rapid degradation through a sustained-release mechanism. To date, various carriers have been used to construct CR systems, including polymer-based pesticide reservoirs such as sodium alginate and polylactic acid (PLA); lipid-based ones such as liposomes; porous inorganic materials such as mesoporous silica and carbon nanotubes; and metal-based materials such as coordination polymers (CPs). CPs are composed of a mixture of organic linkers and metals or metal clusters, and have high specific surface area and porosity, as well as excellent mechanical and chemical stability and customizability. They have been applied to gas adsorption and separation, catalysis, fluorescent labeling in bioimaging and detection, and controlled-release drug delivery systems.

[0004] In the field of controlled-release and delivery of fungicides, CPs (polystyrene-containing compounds) act as antimicrobial agents and carriers through the following mechanisms: generation of antimicrobial free radicals through photocatalysis; penetration and disruption of bacterial membranes by small CP particles, thereby stripping bacteria of their protective barrier; released antimicrobial fungicides participating in antimicrobial activity; and the release of metal ions or ligands with antimicrobial activity upon structural collapse of CPs. Alternaria alternata is a highly adaptable fungal pathogen capable of surviving in diverse environments, including water, soil, and air. Its potent pathogenicity and saprophytic properties enable it to rapidly infect newly harvested strawberries and apples, causing black spot disease and significant economic losses. Tebuconazole (TEB), a triazole fungicide, is renowned for its high efficacy, low toxicity, and broad-spectrum activity, making it effective in treating and preventing plant diseases caused by A. alternata. However, due to its low water solubility, TEB is primarily formulated as an emulsifiable concentrate, which is susceptible to hydrolysis and photolysis in the environment, resulting in low utilization, a short shelf life, and significant environmental damage due to the use of organic solvents. Currently, controlled-release systems for TEB are being developed to address these shortcomings.

[0005] For example, TEB microcapsules (Tebuc@PCN@P@C) prepared by layer-by-layer self-assembly of pectin and chitosan showed dual responses to pH and plant pathogen challenges. By loading tebuconazole into zinc-based metal-organic framework (ZnMOFs) nanoparticles and further encapsulating them with carboxymethyl cellulose (CMC), a fungicide delivery system (Tebu@ZnMOFs@CMC) with dual environmental responses to pH and cellulase was successfully constructed. Unfortunately, the loading rate of TEB in these controlled-release delivery systems is less than 30%, and in practical applications, the formulation requires organic solvents, which also hinders the complete release of the drug. Summary of the Invention

[0006] In view of this, the present invention provides a preparation method and sustained-release antibacterial application of coordination polymerization containing tebuconazole, which has excellent bactericidal effect.

[0007] The specific technical solutions of the present invention are as follows:

[0008] A coordination polymer has zinc as the central metal and ligands of tebuconazole and HCOO-R-COOH, wherein R is selected from at least one of a benzene ring, a heterocycle and an aliphatic group, forming a dual-ligand coordination polymer.

[0009] Preferably, the HCOO-R-COOH is trimesic acid, isophthalic acid, p-aminobenzoic acid, 5-aminoisophthalic acid, biphenyl dicarboxylic acid, 5-hydroxyisophthalic acid, 1,4-naphthalene dicarboxylic acid, fumaric acid, malonic acid, citric acid, succinic acid, glutamic acid, citraconic acid, 2,5-dimethylbenzoic acid, 2,5-dichlorobenzoic acid, 3-chlorobenzoic acid, 3,4-dihydroxyphenylacetic acid, m-hydroxybenzoic acid, 2,5-dihydroxybenzoic acid, 4-phenylbenzoic acid, trimesic acid, naphthoic acid, m-toluic acid, 1,2 ,4-benzenetricarboxylic acid, 1,2,3-benzenetricarboxylic acid, 3,4-dimethylbenzoic acid, 2,5-dimethylphenylacetic acid, pyromellitic acid, oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, dodecanedioic acid, tartaric acid, malic acid, maleic acid, maleic acid, itaconic acid, muconic acid, 3,3',4,4'-biphenyltetracarboxylic acid, 2,6-naphthalene dicarboxylic acid, 4,4'-carbonyldibenzoic acid, 3,3',4,4'-oxybenzene tetracarboxylic acid, and at least one of terephthalic acid.

[0010] A method for preparing a coordination polymer is the method for preparing any of the above-mentioned coordination polymers.

[0011] Preferably, the zinc salt, the HCOO-R-COOH and the tebuconazole are sequentially added to a DMF aqueous solution, heated in an oil bath and stirred to obtain the coordination polymer.

[0012] Preferably, the zinc salt includes at least one of zinc chloride, zinc sulfate and zinc nitrate.

[0013] Preferably, the stirring is carried out at 0-200° C. for 0.1-72 h.

[0014] Preferably, the molar concentration of the zinc salt is 0.01 to 100 mmol / L, the molar concentration of the HCOO-R-COOH is 0.01 to 100 mmol / L, the molar concentration of the tebuconazole is 0.01 to 100 mmol / L, and the molar ratio of the zinc salt, the HCOO-R-COOH and the tebuconazole is (0.01 to 100):(0.01 to 100):(0.01 to 100).

[0015] Use of any of the above coordination polymers, or any of the above preparation methods, in the preparation of pesticides and fungicides.

[0016] Preferably, the coordination polymer is released in an aqueous environment.

[0017] The coordination polymer of the present invention has an ultra-high fungicide loading, excellent fungicidal efficacy, good UV stability, and exhibits dual-responsive release in terms of pH and temperature. The coordination polymer of the present invention not only maintains the antifungal activity of the original fungicide TEB and prolongs the duration of its antifungal effect, but also exhibits improved biosafety compared to TEB.

[0018] The preparation method of the present invention synthesizes a coordination polymer through oil-heat. The method obtains the coordination polymer loaded with pesticides through a one-step reaction, which is shorter than the preparation path of traditional encapsulated pesticides. At the same time, the material obtained by this method can achieve a high loading rate of 73% for the pesticide tebuconazole, and can be widely used.

[0019] The coordination polymer of the present invention can be fully released during application, fully exerts the bactericidal effect, and is environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The following is a brief introduction to the drawings required for use in the embodiments. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without any creative work.

[0021] Figure 1 Schematic diagram of the structure of BUAP3 in Example 1; (a) is the coordination environment of Zn(II), TPA2- and TEB, and (b) is the one-dimensional chain of BUAP3.

[0022] Figure 2 A series of characterizations of BUPA3; (a) the single crystal structure and one-dimensional chain of BUAP3, (b) the P-XRD spectrum (measured and simulated), (c) the particle size distribution, (d) the SEM scan, and (e) the EDS scan.

[0023] Figure 3 The results of BUAP3 determination are as follows; (a) is the FT-IR spectrum of BUAP3, (b) is the TGA spectrum of BUAP3, (c) is the adsorption-desorption curve of N2, and (d) is the BJH (adsorption) pore area and size logarithmic curve.

[0024] Figure 4 (a) is the Zeta potential of BUAP3, Figure 4 (b) XPS spectrum of BUAP3.

[0025] Figure 5 The results of the controlled-release performance test of BUAP3; (a) is the corresponding release of pH, (b) is the corresponding release of temperature, (c) is the cumulative release curve of BUAP3 from TEB in tap water, and (d) is the release kinetic model of BUAP3 in tap water.

[0026] Figure 6 Results of UV resistance test; (a) photodegradation rate of TEB and BUAP3 as a function of time, (b) UV absorption spectra of TEB and TPA, and (c) thermal storage stability analysis of TEB and BUAP3.

[0027] Figure 7 The results of antibacterial effect determination; (a) is the in vitro antibacterial effect of BUAP3, TEB and TPA and EC50, (b) is the growth rate of BUAP3, TEB and TPA, (c) is the inhibition rate of BUAP3 and TEB, (d) is the result of strawberry leaf infection experiment, and (e) is the data statistics of the leaf infection experiment results.

[0028] Figure 8 Results of biosafety assessment; (a) zebrafish survival rate, (b) seed germination rate after fungicide treatment, (c) seed growth length, (d) stem length, and (e) Marc-145 cytotoxicity test results. DETAILED DESCRIPTION

[0029] The technical scheme of the present application will be clearly and completely described below in conjunction with the accompanying drawings and specific embodiments, but it will be understood by those skilled in the art that the following described embodiments are a part of the present application's embodiments, rather than all of the embodiments, and are only used to illustrate the present application, and should not be considered as limiting the scope of the application. It will be understood by those skilled in the art that the specific embodiments of the present invention are modified or some technical features are equivalently replaced, without departing from the spirit of the technical solution of the present invention, and all should be encompassed in the scope of protection of the present invention. In the embodiments, if specific conditions are not indicated, proceed according to the conditions recommended by normal conditions or manufacturers. Reagents used or instruments that do not indicate manufacturers are conventional products that can be purchased commercially.

[0030] A coordination polymer with zinc as the central metal and ligands of tebuconazole and HCOO-R-COOH, wherein R is selected from at least one of a benzene ring, a heterocycle and an aliphatic group, forming a dual-ligand coordination polymer. Zinc is not a heavy metal element and has low biological toxicity. Zinc is a trace element required by organisms and can be moderately absorbed by organisms as a nutrient. The compound HCOO-R-COOH can be trimesic acid, isophthalic acid, p-aminobenzoic acid, 5-aminoisophthalic acid, biphenyldicarboxylic acid, 5-hydroxyisophthalic acid, 1,4-naphthalene dicarboxylic acid, fumaric acid, malonic acid, citric acid, succinic acid, glutamic acid, citraconic acid, 2,5-dimethylbenzoic acid, 2,5-dichlorobenzoic acid, 3-chlorobenzoic acid, 3,4-dihydroxyphenylacetic acid, m-hydroxybenzoic acid, 2,5-dihydroxybenzoic acid, 4-phenylbenzoic acid, trimesic acid, naphthoic acid, m-methylbenzoic acid, 1,2,4-benzenetrimonium chloride, At least one of formic acid, 1,2,3-benzenetricarboxylic acid, 3,4-dimethylbenzoic acid, 2,5-dimethylphenylacetic acid, pyromellitic acid, oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, dodecanedioic acid, tartaric acid, malic acid, maleic acid, maleic acid, itaconic acid, muconic acid, 3,3',4,4'-biphenyltetracarboxylic acid, 2,6-naphthalene dicarboxylic acid, 4,4'-carbonyldibenzoic acid, 3,3',4,4'-oxybenzene tetracarboxylic acid, and terephthalic acid, preferably terephthalic acid. The coordination polymer has a size ranging from nanometers to micrometers, which provides better adhesion after spraying.

[0031] The reactants of the coordination polymer can be reacted in an alcohol reagent or a DMF aqueous solution.

[0032] The preparation method of the coordination polymer may include the following steps:

[0033] (1) DMF and water are mixed in a molar ratio of (1-100): (100-1) to obtain a DMF aqueous solution.

[0034] (2) A zinc salt, terephthalic acid, and tebuconazole are sequentially added to a DMF aqueous solution and mixed uniformly. The zinc salt comprises at least one of zinc chloride, zinc sulfate, and zinc nitrate, preferably zinc chloride. Stirring is performed in an oil bath at 0 to 200°C for 0.1 to 72 hours. The heating temperature may be 0°C, 80°C, 90°C, 100°C, 120°C, 200°C, etc. The stirring time may be 0.1 hour, 0.5 hour, 3 hours, 6 hours, 72 hours, etc. The stirring speed is 120 rpm. BUAP3 crystals grow and precipitate rapidly.

[0035] The preparation method of the coordination polymer may further comprise the following steps:

[0036] (1) DMF and water are mixed in a molar ratio of (1-100): (100-1) to obtain a DMF aqueous solution.

[0037] (2) A zinc salt, terephthalic acid, and tebuconazole are sequentially added to a DMF aqueous solution and mixed uniformly. The zinc salt comprises at least one of zinc chloride, zinc sulfate, and zinc nitrate, preferably zinc chloride. The reaction is carried out at 80-120°C for 48-96 hours, and the temperature is preferably 120°C. BUAP3 crystals grow freely. The reaction product is washed with ethanol and then water, and dried at room temperature to obtain a millimeter-sized white solid product.

[0038] The products obtained by the above preparation methods are of the same type and have the same characteristics.

[0039] Example 1

[0040] A coordination polymer with zinc as the central metal and TEB and terephthalic acid as ligands, forming a double ligand coordination polymer named BUAP3, whose structure is as follows Figure 1 .

[0041] Example 2

[0042] 1. Materials and Methods

[0043] 1.1. Materials

[0044] Zinc chloride, H2TPA, TEB, TEB emulsion, and sodium N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonicacid) (HEPES) were provided by J&K Scientific Co., Ltd. N,N-dimethylformamide (DMF), methanol (chromatographic grade), and tetrahydrofuran (THF) were provided by Aladdin Chemical Reagent Co., Ltd. Dulbecco's Modified Eagle Medium (DMEM) and potato dextrose agar (PDA) were purchased from Qingdao Haibo Biotechnology Co., Ltd. (Qingdao, China). Cell Counting Kit-8 (CCK-8) was purchased from Sun Biotechnology Co., Ltd. (Beijing, China). Alternaria alternata and Marc-145 cells were provided by the Key Laboratory of Urban Agriculture of North China (Beijing, China). Ultrapure water was provided by Milli- Produced using an EQ7000 ultrapure water purification system. Adult zebrafish were purchased from the Yushen Aquarium in Beijing.

[0045] 1.2. Instrumentation and characterization

[0046] The MAGNA-560 spectrometer was used to measure the wavelengths in the range of 400-4000 cm -1Fourier transform infrared (FT-IR) spectra of the CP were obtained. Powder X-ray diffraction (P-XRD) spectroscopy was performed on a Bruker D8 focused diffractometer using a copper target tube and a graphite monochromator. P-XRD spectra were simulated using Mercury single crystal X-ray diffraction (SCXRD) data and Mercury software. Thermogravimetric analysis (TGA) was performed on a Rigaku Thermo+ EVO2TG-DTA8121 analyzer, heating from room temperature to 800°C under a nitrogen atmosphere. The specific surface area of the CP was determined using a BSD-PM1 microporous analyzer. The specific surface area was measured using the BET method. A SWC-LGE self-cooling freezing point tester was used to control the temperature of the sustained-release system. Elemental analysis (EA) was performed using a PerkinElmer 240C analyzer (for C, H, and N). High-performance liquid chromatography (HPLC) detection was performed using an Agilent 1200 series HPLC system (Agilent, USA) equipped with a G1314B detector (Agilent, USA) and a ZORBAX Eclipse XDB-C18 (4.6 mm × 150 mm, 5 μm).

[0047] 1.3.[Zn4(TEB)8(TPA)4] n Preparation

[0048] Synthesis of millimeter-scale large-sized BUAP3: A Teflon-lined autoclave (100 mL) was charged with zinc chloride (4 equiv, 0.2 mmol, 0.0272 g), H2TPA (1 equiv, 0.05 mmol, 0.0083 g), TEB (1 equiv, 0.05 mmol, 0.0153 g) and a mixed solvent (DMF: water volume = 1:1, 4 mL). The resulting mixture was kept at 80 °C for 48 hours. Then, the white crystalline solid was filtered out from the solvent and washed with water and methanol. The solid was then dried under open air conditions to obtain 0.0113 g of the final product. The synthesized crystals were used for single crystal X-ray diffraction analysis. FT-IR (neat, ν / cm -1 ): 3567(w), 1566(s), 1522(s), 1504(m), 1385(s), 1277), 1128), 1086) , 1013, 992), 839(s), 751(s), 696(w), 669(s), 657(s), 530(s), 401(s).

[0049] Synthesis of small BUAP3 particles: To further optimize the reduction of BUAP3 particle size, a 100 mL flask was charged with zinc chloride (1 eq., 0.2 mmol, 0.0272 g), H2TPA (1.25 eq., 0.25 mmol, 0.0415 g), TEB (2.5 eq., 0.5 mmol, 0.153 g), and a mixed solvent (DMF:water = 1:1 by volume, 20 mL). The resulting mixture was rapidly stirred in an 80°C oil bath at 120 rpm for 6 h, and the final product was collected for subsequent experiments.

[0050] 1.4. Fungicide release analysis

[0051] The concentration of TEB was determined by HPLC under the following conditions: injection volume of 10 μL for each sample, column temperature maintained at 30°C, mobile phase of methanol and water (80:20, v / v), and effluent flow rate maintained at 1.0 mL / min. The UV detector was set at 220 nm.

[0052] 1.4.1. Environmentally responsive release

[0053] pH-responsive release: CP (80 mg) and HEPES buffer (80 mL) were dissolved in ultrapure water (80 mL) to prepare BUAP3 suspensions. The solutions were then stirred at room temperature, and 2 mL of each suspension was centrifuged after a fixed time, and an equal volume of fresh buffer was added.

[0054] Temperature-responsive release: Follow the above procedure, except that the experiment was maintained at different temperatures (10, 20, and 30°C) in HEPES buffer (pH 7). TEB concentration was determined by HPLC. Three replicates were performed for each condition.

[0055] 1.4.2. Cumulative release of TEB

[0056] BUAP3 (50 mg) was dispersed in 1500 mL of tap water and stirred. 2 mL samples were taken periodically, centrifuged, and analyzed by liquid chromatography. The 2 mL tap water sample and the centrifuged solid mixture were then added to the suspension. The TEB concentration released from the BUAP3 suspension was determined by HPLC, and the cumulative release rate (CRR) of TEB was calculated using the following equation:

[0057]

[0058] Where Mt is the amount of TEB released at each sampling time, t is the sampling time, and M is the theoretical amount of TEB in BUAP3.

[0059] Release kinetics model

[0060] The BUAP3 release kinetics were fitted using the zero-order, first-order, Higucci, and Ritger-Peppas models using the equations as follows:

[0061]

[0062] Wherein, t is the amount of drug released at time t, M∞ is the maximum amount of drug released at infinite time, k is the rate constant of the bactericidal agent, and n is the diffusion exponent [Fickian diffusion (n≤0.43), non-Fickian diffusion, or non-Fickian diffusion (0.43 <n<1.0)]。

[0063] 1.5. Stability test

[0064] 1.5.1. Determination of UV resistance

[0065] 16.5 mg of BUAP3 (12 mg based on TEB) was added to 50 mL of water to obtain a suspension concentration of 240 mg / L (based on TEB). Simultaneously, a 240 mg / L TEB solution was prepared by diluting a commercial emulsion with water. A simulated decomposition process was performed at room temperature using a 365 W mercury lamp as the irradiation source at a wavelength of 254 nm. Aliquots (1 mL) were taken at regular intervals and diluted with an equal volume of THF to completely dissolve the BUAP3 particles.

[0066] 1.5.2. Thermal storage stability determination

[0067] A 100 mg sample of BUAP3 was weighed and stored at 54° C. (±2° C.) for 14 days. The sample was then dissolved in THF by HPLC to determine the residual amount of TEB in BUAP3.

[0068] 1.6. Biological activity studies

[0069] 1.6.1. In vitro antifungal assay

[0070] The fungicidal activity of BUAP3 against Alternaria alternata (provided by the Key Laboratory of Urban Agriculture, North China) was determined using the growth rate method and compared with TEB. 10 mg of BUAP3 suspension was stirred in 100 mL of water, 2 mL of the suspension was taken, and the TEB concentration in the supernatant was determined. Before the antifungal test, BUAP3 suspension (concentration determined based on TEB), BUAP3 suspension (concentration based on clear solution free of TEB), TEB solution, and TPA solution were used to prepare potato dextrose agar (PDA) at concentrations of 0, 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 mg / L, respectively. Then, for each article, a 5 mm diameter bacterial cake was inoculated in the center of a 60 mm culture dish and incubated at 28°C until the fungal colony approached the edge of the culture plate. The colony diameter was determined by the intersection method, and the inhibition rate was calculated according to the equation:

[0071]

[0072] Where G1 is the diameter of the fungus cake in the control group, G x is the diameter of the fungus cake in the treatment group, and G0 is the initial diameter of the fungus cake.

[0073] 1.6.2. Potted plant experiments

[0074] The antibacterial activity of BUAP3 and TEB against strawberry black spot disease was evaluated. Strawberries were cultured in a greenhouse at 28°C and 80% humidity. A BUAP3 suspension (50.0 mg / L based on the active ingredient of TEB) or a TEB solution (50.0 mg / L) was sprayed onto strawberry leaves, which were then inoculated with a 5 mm diameter fungal cake and cultured at 28°C. The control group was treated with an equal amount of water. After a certain period of time, the spot area of all test leaves was determined. The results were recorded after 2 weeks of culture at 28°C. The lesion area on the leaves was calculated using ImageJ software.

[0075] 1.7. Safety evaluation of wheat seed germination

[0076] Aqueous TEB solutions or suspensions of BUAP3 (based on the active ingredient) were prepared at concentrations of 25.0, 50.0, and 100.0 mg / L. Wheat seeds (purchased from the Beinong Market) were soaked in water for two days, dried, and then sprayed with tap water (control), the TEB solution, or the BUAP3 suspension. The seeds were then transferred to a dark chamber and incubated at 25°C for seven days. Stem length and germination rate were recorded and calculated. Each experiment was repeated three times, and the results were averaged.

[0077] 1.8. Acute toxicity to adult zebrafish

[0078] Acute toxicity studies of BUAP3 and TEB on adult zebrafish were conducted in accordance with Test Guide No. 203. Adult zebrafish, 2.5 (± 0.5) cm in diameter, were placed in chlorine-free tap water at 24°C with a 10 / 14 dark / light cycle for one week. Aquaria (4 L, 20 cm × 16 cm × 13 cm) were then filled halfway with TEB solute or BUAP3 suspension at concentrations of 0 (control), 1.0, 2.0, 4.0, 6.0, or 8.0 mg / L. Ten zebrafish were housed in each aquarium for 96 hours, and the LC50 (50% lethal concentration) was calculated using GraphPad Prism 9.0.

[0079] 1.9. Cytotoxicity Assay

[0080] The CCK-8 method was used to evaluate the cytotoxic effects of BUAP3 and TEB on MARC-145 cells. MARC-145 cells were propagated in Dulbecco's modified Eagle's medium (DMEM) and cultured in 96-well plates in a humidified atmosphere containing 95% air and 5% carbon dioxide at 37°C for 2 to 3 days. Then, BUAP3 or TEB was introduced into the cell culture system to obtain concentrations of 0 (control), 3.175, 6.25, 12.5, 25, and 50 (based on the total active ingredient) and cultured for 24 hours. After incubation with the test component, MARC-145 cells were further cultured for 24 hours after the addition of CCK-8 reagent. After the culture period, the absorbance of MARC-145 cells at 450 nm was measured using a microplate reader. Cell viability was calculated using the equation software:

[0081]

[0082] Where A is the absorbance of the experimental group (including medium, cells, test materials, and CCK-8 solution), B is the absorbance of the control group (including medium, cells, and CCK-8 solution), and C is the absorbance of the blank group (including medium and CCK-8 solution).

[0083] 2. Results and Discussion

[0084] Synthesis and structure

[0085] BUAP3 was synthesized from H2TPA, zinc sulfate and TEB under hydrothermal conditions. The structure of BUAP3 was revealed by single crystal X-ray diffraction analysis. The asymmetric unit consists of four crystal-independent Zn(II) ions, four TPA 2- and 8 TEB ligands, such as Figure 2 As shown in (a), half of the ions and molecules are disordered in two parts with an occupancy of 0.5. The central Zn(II) ion is coordinated with 4 imidazole moles of N in TEB, TPA 2- The four O atoms of the two carboxylic acid groups are coordinated. Zn(II) ions and TPA 2- The ions alternate with each other to form a zigzag one-dimensional chain, with two TEBs acting as end chains, bound next to each metal center. [Zn4(TEB)8(TPA)4] n The structure shows that the loading rate of TEB in the material is 72.85%.

[0086] 2.2. Characterization

[0087] Figure 2(a) is the powder X-ray diffraction (P-XRD) test spectrum of BUAP3 and the simulant. The P-XRD spectrum is consistent with the simulation results, indicating that the purity of BUAP3 material is high. In further research, the morphology of BUAP3 was observed by transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDS), as shown in Figure 2. Figure 2 (c) and Figure 2 (d) As shown. The synthesized BUAP3 is a cuboid crystal with an average size of about 5 μm. Figure 2 (b) shown. Figure 2 (e) is the EDS elemental spectrum of BUAP3 particles, indicating the presence and uniform distribution of C, N, O, Cl, and Zn elements in BUAP3 particles.

[0088] The Fourier transform infrared (FT-IR) spectrum of BUAP3 showed several characteristic peaks that provided insights into the functional groups and bonding interactions within the framework, such as Figure 3 (a) The asymmetric stretching vibration of the carbon group in the TPA2-carboxylic acid group occurs at 1573 cm -1 The symmetrical stretching vibration occurs at 1375 cm -1 There is no obvious peak around 1680cm-1, which indicates the lack of free carboxylic acid groups, confirming that the carboxylic acid groups are fully coordinated with the Zn(II) center. -1 The peak at 1510 cm is due to the stretching vibration of the CO bond. -1 The peak belongs to the vibration absorption of -C=N in the heterocycle, at 679cm -1 The peak at 1379 cm is derived from the vibration absorption of C-Cl bond and -1 Corresponding to the stretching vibration of -OH in TEB.

[0089] Thermogravimetric analysis (TGA) of BUAP3 Figure 3 (b) The thermal degradation-induced weight loss of TEB and BUAP3 occurs at temperatures around 250°C and 290°C, respectively. The weight loss curve for BUAP3 decreases abruptly from 290°C to 350°C, with an inflection point observed above 350°C after curing, likely due to the onset of TPA2 ligand decomposition. With increasing temperature, a relatively stable sample weight is observed above 550°C, with the sample weight remaining decreasing from 15.2% to 11.9%.

[0090] Figure 3 (c) shows the N2 adsorption-desorption isotherm and BUAP3. There is a hysteresis line between the relative pressure (P / P0) of 0.2 and 1.0, so the isotherm can be classified as type IV. Barrett-Joyner-Halendar (BJH) shows the pore size distribution, such as Figure 3As shown in (d), the average pore size calculated according to the BJH model is 10.1 nm, proving that BUAP3 exhibits a mesoporous structure, and the specific surface area is 1.58 m2 / g according to the multi-point method.

[0091] Figure 4 (a) Shows the zeta potential of BUAP3 suspensions at different pH values. At pH 5, 7, and 9, the average surface potential of the suspensions was 5.7, -22.4, and -34.8 mV, respectively. At pH 7, the absolute value of the BUAP3 surface charge was the highest, indicating increased electrostatic repulsion between particles, making the material more easily dispersed in aqueous environments.

[0092] The chemical composition and element valence state of BUAP3 material were evaluated by XPS method. Figure 4 The XPS spectrum in (b) shows that the elements present in BUAP3 are Zn, C, O, N, and Cl. In the high-resolution spectrum, the peak of C 1s at 284.8 eV belongs to CC; the peak of O 1s at 532.6 eV comes from CO; for N1s and Cl 2p, the N and Cl elements of TEB in BUAP3 are detected, respectively, providing evidence for the successful loading of TEB.

[0093] Controlled Release of BUAP3

[0094] Table 1 TEB release kinetic equations of BUAP3 in water environment.

[0095] Functional model Fitting function <![CDATA[R 2 ]]> Zero Order y=1.34t+32.99 0.81 First level <![CDATA[y=95.48(1-e -0.12t )]]> 0.94 Shiguchi <![CDATA[y=12.29t 0.5 +14.04]]> 0.95 Ritger Peppas <![CDATA[y=25.35t 0.35 ]]> 0.97

[0096] The cumulative release test of BUAP3 TEB was carried out in water environment, e.g. Figure 5 (c) As shown. Under stirring conditions, TEB was released rapidly within the first 40 h, and after the release at this node, its release amount reached about 98%. This shows that BUAP3 can almost achieve complete release of TEB. The remaining 2% that can be released may be due to the presence of a small amount of impurities in the material and the measurement error caused by the experimental instrument. At the same time, the release behavior of BUAP3 in tap water was fitted using four common fungicide release kinetic models, such as Figure 5 (d) As shown in Table 1, R 2 The values were 0.81, 0.94, 0.95, and 0.97, respectively. In other words, the release behavior of BUAP3 in tap water is more consistent with the Ritger-Peppas model. The release exponent n in this model is 0.35 < 0.45, indicating that tebuconazole is released by Fickian diffusion (Case I), that is, its release conforms to the diffusion process of the polymer.

[0097] Figure 5 (a) shows the real-time release concentration of TEB from BUAP3 in aqueous environments at pH values of 5, 7, and 9. At pH 9, TEB was rapidly released from 0 to 10 hours, but the release rate gradually slowed, ultimately stabilizing at a concentration of around 30 mg / L around 25 hours. At pH 5, TEB was rapidly released within the first 5 hours, then slowly released for approximately 25 hours before the concentration stabilized at around 30 hours. At pH 7, TEB was slowly released until around 45 hours, when the concentration stabilized. In summary, BUAP3 releases both acidic and alkaline reactions, with alkaline conditions reacting more rapidly and acidic conditions reacting more steadily. The surface potential of PDPC3 also changes significantly with changes in the pH of the external liquid environment. This phenomenon alters the surface charge state of BUAP3 particles, disrupting the original coordination electrostatic adsorption equilibrium between BUAP3 particles and the TEB biocide.

[0098] The release curves under different temperature conditions (i.e. 10, 20, 30°C to simulate day and night) are as follows: Figure 5 (b) The release rate of TEB was positively correlated with temperature, and the TEB concentration remained stable for approximately 30 hours. The results showed significant differences due to temperature variations. Low temperatures slowed molecular motion, and the TEB biocide molecules in the coordinated form of BUAP3 particles gained insufficient energy, making it difficult to escape the constraints of the particles and the coordination bonds, resulting in slow TEB biocide release into the solution. In summary, BUAP3 exhibits a temperature-responsive release effect.

[0099] 2.4. UV resistance

[0100] Figure 6 (a) Degradation rates of BUAP3 and TEB after different light exposure times. TEB rapidly degraded at the beginning of light exposure, then stabilized, reaching 95% degradation at 3.5 hours. Meanwhile, the degradation rate of BUAP3 was only approximately 35% at 3.5 hours, indicating that BUAP3 significantly improved the photostability of loaded TEB.

[0101] Figure 6 (b) UV absorption of TPA and TEB at 10 mg / L. There are many factors that can be considered to improve the UV stability of BUAP3. Ligand stability can often be enhanced by incorporating high-density materials. Compared to TEB, H2TPA exhibits higher UV absorption at wavelengths of 215–275 nm. Therefore, protection of TEB from radiation-induced decomposition by the TPA ligand may be another factor contributing to the greater stability of TEB in BUAP3.

[0102] Figure 6(c) Exploring the thermal storage stability of BUAP3, the residual TEB content in BUAP3 changes over time at an ambient temperature of 54°C. By day 14, the remaining TEB content still reached approximately 95% of the initial amount, demonstrating that long-term high-temperature storage does not significantly affect the stability of BUAP3. Furthermore, in the TGA test, the weight of BUAP3 only began to decrease after 270°C. In summary, BUAP3 exhibits excellent thermal stability.

[0103] 2.5. Inhibitory effect on Alternaria alternata

[0104] The antibacterial activity of BUAP3 against strawberry black spots was evaluated by the antibacterial activity of TEB against strawberry black spots. Figure 7 (a) shows the inhibitory effects of BUAP3, TEB and TPA at different concentrations. The antifungal activities of all three compounds showed a concentration-dependent enhancement. Figure 7 As shown in (c), the EC50 value of BUAP3 was 1.98±0.28 mg / L, while that of TEB was 2.00±0.32 mg / L, indicating that the antifungal effect of BUAP3 was slightly better than that of TEB. Figure 7 The histogram in (b) clearly shows that TPA has a significant antifungal effect when the concentration reaches 2 mg / L. It can be inferred that the slightly enhanced antifungal effect of BUAP3 relative to TEB is due to the addition of TPA2 to BUAP3.

[0105] Pot experiments were conducted to further evaluate the antibacterial activity of BUAP3 against strawberry black spots. Figure 7 As shown in (d) and (e), the BUAP3 granule group had a smaller lesion area on strawberry leaves at the same time point compared to the TEB group and the blank group, indicating that BUAP3 has good antifungal activity in living plants. Over time, when lesions in the TEB group began to appear and continued to expand, the BUAP3 granule group continued to have good antifungal activity.

[0106] 2.6. Biosafety assessment

[0107] In practical applications, fungicides inevitably enter the aquatic environment due to factors such as leaching, thereby affecting the safety of non-target organisms. In the risk assessment of fungicides in aquatic environments, zebrafish are an important biological specimen for acute toxicity testing of fungicides in fish. Figure 8 (a) shows the acute toxicity of TEB and BUAP3 to zebrafish during the experimental period of 24 to 96 h. At the same concentration between groups, the LC 50The values of BUAP3 and BUAP3-immobilized TEBs reached 3.3 mg / L, while the legality of BUAP3 particles at a concentration of 10 mg / L was approximately 7% and 13%, respectively. This indicates that BUAP3-immobilized TEBs have good biosafety.

[0108] TEB can inhibit wheat germination by disrupting the redox homeostasis of wheat. Figure 8 (b), (c), and (d) show the germination rate and stem length of wheat seeds after treatment with TEB and BUAP3. Compared to the untreated blank control, both the TEB and BUAP3 treatments showed inhibitory effects on wheat seed germination. At the same concentration, the BUAP3 treatment increased the germination rate of wheat seeds by approximately 30% compared to the TEB treatment at different pesticide concentrations. Furthermore, the stem length of wheat seeds in the BUAP3 treatment group was approximately 2 cm longer than that in the TEB treatment group. These results indicate that BUAP3 is significantly less toxic to wheat germination than TEB.

[0109] The toxicity of BUAP3 to Marc-145 cells is as follows Figure 8 (e) The results showed that both BUAP3 and TEB exhibited concentration-dependent cytotoxicity. However, at the same concentration, cell viability in the BUAP3 group was greater than that in the TEB group. In particular, at a concentration of 50 mg / L, cells in the TEB group barely survived, while cells in the BUAP3 group maintained a high survival rate of nearly 50%.

[0110] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some or all of the technical features therein, and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A coordination polymer, characterized in that Zinc is used as the central metal, and the ligands are tebuconazole and HCOO-R-COOH, wherein R is selected from at least one of a benzene ring, a heterocycle and an aliphatic group, to form a dual-ligand coordination polymer.

2. The coordination polymer according to claim 1, characterized in that The HCOO-R-COOH is trimesic acid, isophthalic acid, p-aminobenzoic acid, 5-aminoisophthalic acid, biphenyl dicarboxylic acid, 5-hydroxyisophthalic acid, 1,4-naphthalene dicarboxylic acid, fumaric acid, malonic acid, citric acid, succinic acid, glutamic acid, citraconic acid, 2,5-dimethylbenzoic acid, 2,5-dichlorobenzoic acid, 3-chlorobenzoic acid, 3,4-dihydroxyphenylacetic acid, m-hydroxybenzoic acid, 2,5-dihydroxybenzoic acid, 4-phenylbenzoic acid, trimesic acid, naphthoic acid, m-toluic acid, 1,2,4 at least one of trimellitic acid, 1,2,3-benzenetricarboxylic acid, 3,4-dimethylbenzoic acid, 2,5-dimethylphenylacetic acid, pyromellitic acid, oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, dodecanedioic acid, tartaric acid, malic acid, maleic acid, maleic acid, itaconic acid, muconic acid, 3,3',4,4'-biphenyltetracarboxylic acid, 2,6-naphthalene dicarboxylic acid, 4,4'-carbonyldibenzoic acid, 3,3',4,4'-oxybenzene tetracarboxylic acid, and terephthalic acid.

3. A method for preparing a coordination polymer, characterized in that: The invention relates to a method for preparing the coordination polymer according to claim 1 or 2.

4. The preparation method according to claim 3, characterized in that The zinc salt, the HCOO-R-COOH and the tebuconazole are sequentially added into a DMF aqueous solution, heated in an oil bath and stirred to obtain the coordination polymer.

5. The preparation method according to claim 3, characterized in that The zinc salt includes at least one of zinc chloride, zinc sulfate and zinc nitrate.

6. The preparation method according to claim 3, characterized in that Stir at 0-200°C for 0.1-72h.

7. The preparation method according to claim 3, characterized in that The molar concentration of the zinc salt is 0.01 to 100 mmol / L, the molar concentration of the HCOO-R-COOH is 0.01 to 100 mmol / L, the molar concentration of the tebuconazole is 0.01 to 100 mmol / L, and the molar ratio of the zinc salt, the HCOO-R-COOH and the tebuconazole is (0.01 to 100):(0.01 to 100):(0.01 to 100).

8. Use of the coordination polymer according to claim 1 or 2, or the preparation method according to any one of claims 3 to 7, in the preparation of pesticides and fungicides.

9. The use according to claim 8, characterized in that The coordination polymer is released in an aqueous environment.

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