Pesticide slow-release agent, preparation method and application thereof

Abstract

This invention belongs to the field of pesticide formulation technology, and relates to a pesticide sustained-release agent, its preparation method, and its application. The sustained-release agent is a hydrogel with a three-dimensional porous network structure formed by the self-assembly of glycyrrhizic acid or glycyrrhetinic acid and pyraclostrobin through intermolecular non-covalent interactions. The porosity is 70%-90%, and the average pore size is 2-5 μm. This invention co-assembles the fungicide pyraclostrobin and glycyrrhizic acid based on natural products to form a stable three-dimensional supramolecular hydrogel sustained-release agent, achieving controlled release and efficient utilization of the drug, exhibiting good fungicidal activity and biosafety. Furthermore, this carrier-free drug delivery system can effectively avoid the environmental harm caused by formulation residues, and is suitable for the practical needs of green agriculture and sustainable plant protection, possessing significant application value and promising prospects for promotion.

Description

Technical Field

[0001] This invention belongs to the field of pesticide formulation technology, and relates to a pesticide slow-release agent, its preparation method and application. Background Technology

[0002] Pesticides play a vital role in modern agricultural production, but traditional pesticides suffer from problems such as low utilization rates, severe environmental pollution, easy photodegradation, and easy runoff. Statistics show that the actual utilization rate of pesticides is typically less than 10%, and large amounts of pesticide residues pose a threat to ecosystems and human health. To improve pesticide utilization efficiency, reduce application frequency, and minimize environmental pollution, pesticide slow-release technology has emerged.

[0003] In recent years, nanotechnology and supramolecular self-assembly strategies have provided new ideas for the development of pesticide sustained-release agents. In particular, co-assembly systems based on natural products combine pesticide molecules with natural active ingredients through non-covalent interactions such as π-π stacking, hydrogen bonding, and hydrophobic interactions, forming carrier-free, biodegradable, and environmentally friendly sustained-release systems. These systems not only avoid the potential toxicity of synthetic carriers but also leverage the bioactivity of natural products to achieve synergistic effects with pesticides.

[0004] Azoxystrobin is a broad-spectrum, highly effective, and low-toxicity fungicide, but its poor water solubility, rapid photodegradation, and short duration of action limit its application. Achieving controlled release and efficient utilization of the drug has significant application value and promising prospects for wider application. Summary of the Invention

[0005] The main objective of this invention is to overcome the deficiencies in the prior art and provide a pesticide slow-release agent, its preparation method, and its application.

[0006] To achieve the above objectives, the specific technical solution is as follows: This invention provides a pesticide slow-release agent, which is a hydrogel with a three-dimensional porous network structure formed by the self-assembly of glycyrrhizic acid or glycyrrhetinic acid and pyraclostrobin through intermolecular non-covalent interactions. The porosity is 70%-90% and the average pore size is 2-5 μm.

[0007] Azoxystrobin is a broad-spectrum, highly effective, and low-toxicity fungicide, but its poor water solubility, rapid photolysis, and short duration of action limit its application. Glycyrrhizic acid, a natural triterpenoid saponin, possesses excellent amphiphilicity, gelling properties, and biocompatibility. This invention uses natural product glycyrrhizic acid as a building block, co-assembled with azoxystrobin through hydrogen bonds, π-π stacking, and hydrophobic interactions to form a stable three-dimensional hydrogel network, creating a carrier-free pesticide slow-release agent. This agent has excellent drug loading space and diffusion channels, enabling intelligent response to the pH value of the external environment. It exhibits a faster release rate in acidic environments (pH=3-5), making it suitable for applications requiring rapid onset of action. Under neutral conditions, the release behavior is more stable and prolonged, significantly extending the duration of the antibacterial effect. This can be flexibly adjusted according to actual application scenarios, significantly improving pesticide utilization efficiency and control efficacy.

[0008] The pesticide slow-release agent of this invention not only effectively overcomes the technical bottlenecks of azoxystrobin's poor water solubility, easy photodegradation, and short effective period, but also avoids the introduction of any synthetic polymer carrier, thus avoiding secondary pollution and biotoxicity risks. It has comprehensive advantages such as environmental friendliness, controlled release, high utilization rate and good biocompatibility, and is suitable for the actual needs of green agriculture and sustainable plant protection.

[0009] Further, the mass ratio of glycyrrhizic acid to azoxystrobin is 30-80:1-6, preferably 40:1; or, the mass ratio of glycyrrhetinic acid to azoxystrobin is 30-80:1-6, preferably 40:1.

[0010] This invention optimizes the mass ratio of glycyrrhizic acid or glycyrrhetinic acid to pyraclostrobin, thereby controlling the crosslinking density and pore structure of the hydrogel network. When the mass ratio of glycyrrhizic acid or glycyrrhetinic acid to pyraclostrobin is within the above-mentioned range, a three-dimensional network structure can be stably formed, which not only ensures the efficient loading of pyraclostrobin, but also provides a reasonable diffusion path for drug release.

[0011] When the preferred 40:1 ratio is used, the resulting hydrogel network exhibits the most suitable degree of cross-linking and pore size distribution. The gel presents a uniform three-dimensional porous network structure with an average pore size of 4.06 μm, clear interfaces, and no phase separation, providing an ideal carrier structure for the loading and release of drug molecules. At the same time, it ensures that the drug release rate reaches an ideal balance, that is, rapid release in an acidic environment to quickly exert its efficacy, and slow and sustained release in a neutral environment to maintain a long-term antibacterial effect, thereby maximizing the bioavailability and control duration of pesticides. Meanwhile, it ensures the structural stability and mechanical properties of the hydrogel itself, facilitating storage, transportation, and application in practical applications.

[0012] The present invention also provides a method for preparing the above-mentioned pesticide slow-release agent, comprising the following steps: (1) Glycyrrhizic acid or glycyrrhetinic acid and pyraclostrobin are dissolved in an organic solvent and then evaporated by rotary evaporation to form a film; (2) Add hydration medium, disperse by ultrasonication, and form a stable hydrogel by cyclic heating-cooling.

[0013] The preparation method of the present invention is simple to operate, requires no complicated equipment or harsh reaction conditions, and is easy to scale up; the entire preparation process does not introduce any toxic or harmful reagents, is green and environmentally friendly, and conforms to the concept of sustainable development.

[0014] Furthermore, the mass ratio of glycyrrhizic acid or glycyrrhetinic acid to pyraclostrobin is 30-80:1-6, preferably 40:1.

[0015] Further, in step (1), the organic solvent is selected from one or more of methanol, ethanol, and acetonitrile, with methanol being preferred.

[0016] This invention uses methanol as an organic solvent, which can effectively dissolve glycyrrhizic acid and azoxystrobin to form a homogeneous solution system, providing a good foundation for the subsequent rotary evaporation film formation step and ensuring that the two components are uniformly distributed in the film. At the same time, methanol has a low boiling point and is easily volatilized and removed during rotary evaporation, without leaving any residue in the film, thus avoiding adverse effects on the subsequent hydrogel formation and performance. Moreover, its volatility makes the film formation process highly efficient, which helps to improve the overall preparation efficiency.

[0017] Further, in step (2), the hydration medium is selected from one or more of water and phosphate buffer, preferably water.

[0018] This invention uses water as the hydration medium. Water, being a polar solvent, can form excellent interactions with the polar groups (such as carboxyl and hydroxyl groups) in glycyrrhizic acid molecules, promoting the swelling of glycyrrhizic acid and the construction of a gel network during hydration. This is beneficial for forming a structurally stable and high-performance hydrogel. Furthermore, using water as the medium eliminates the need for complex solvent separation or purification steps, further simplifying the preparation process, reducing production energy consumption and costs, and aligning with the trend of green chemistry.

[0019] Furthermore, the amount of glycyrrhizic acid or glycyrrhetinic acid dissolved in each 10 ml of the organic solvent is 60-160 mg; the volume ratio of the hydration medium to the organic solvent is 1:(2-6), preferably 1:5.

[0020] Furthermore, in step (2), the power of the ultrasound is 50-200 W, and the duration of the ultrasound is 5-15 min.

[0021] This invention utilizes ultrasound to assist in the formation of uniform and stable nano- or micron-scale structures (such as nanomicelles) and improve the dispersibility of active ingredients. The aforementioned power and ultrasound duration help to uniformly disperse functional nanoparticles (such as drug-loaded micelles) into the gel matrix, prevent agglomeration, and ensure the uniformity and stability of the final product.

[0022] Further, in step (2), the number of cycles is 1-5 times, the heating temperature is 40-60℃, the heating time is 5-30 min, and the cooling temperature is room temperature.

[0023] This invention uses cyclic heating-cooling to induce and regulate molecular self-assembly, ultimately forming a stable three-dimensional gel network. The aforementioned cyclic heating-cooling conditions help to obtain composite gels with superior performance (such as mechanical strength and sustained-release properties).

[0024] The present invention further provides the application of the above-mentioned pesticide slow-release agent in inhibiting agricultural pathogens.

[0025] Furthermore, the pathogens are *Gyromitra esculenta*, *Early Blight*, *Small Spot*, *Powdery Mildew*, and *Downy Mildew*.

[0026] Compared with the prior art, the present invention has the following significant advantages: This invention addresses the problems of low effective utilization, significant harm to non-target organisms, and environmental pollution caused by carrier residues in traditional pesticide formulations. It co-assembles the fungicides azoxystrobin and glycyrrhizic acid based on natural products to form a stable three-dimensional supramolecular hydrogel sustained-release agent, achieving controlled release and efficient utilization of the drug. It exhibits good fungicidal activity and biosafety. More importantly, this carrier-free drug delivery system can effectively avoid the environmental harm caused by formulation residues, making it suitable for the practical needs of green agriculture and sustainable plant protection, and has significant application value and promotion prospects. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0028] Figure 1 This is a scanning electron microscope image of the pesticide slow-release agent of the present invention; Figure 2 Powder X-ray diffraction pattern of the pesticide slow-release agent of this invention; Figure 3 This is the Fourier transform infrared spectrum of the pesticide slow-release agent of this invention; Figure 4 These are the rheological test results of the pesticide slow-release agent of this invention; Figure 5 This is a contact angle test diagram of the pesticide slow-release agent of the present invention; Figure 6This is a graph showing the in vitro antibacterial effect of the pesticide slow-release agent of the present invention under different concentrations; Figure 7 This is a diagram showing the in vitro antibacterial effect of the pesticide slow-release agent of the present invention under different pH conditions; Figure 8 This is a diagram illustrating the effect of the GA-AZOX sustained-release gel of this invention in inhibiting early blight of tomato and small spot disease of corn; (a) Tomato early blight pathogen; (b) Corn leaf spot pathogen.

[0029] Figure 9 This is the result of the tomato live antibacterial experiment of the present invention; Figure 10 This invention relates to the effects of the pesticide slow-release agent on tomato seed germination and growth. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0031] Unless otherwise specified in the embodiments of the present invention, the techniques or conditions described in the literature in this field or the product instructions shall be followed; if the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased through legitimate channels.

[0032] Example This embodiment provides a pesticide slow-release agent, which is a hydrogel with a three-dimensional porous network structure formed by the self-assembly of glycyrrhizic acid and pyraclostrobin through intermolecular non-covalent interactions. Figure 1 As shown in the scanning electron microscope (SEM) image, the hydrogel exhibits a uniform three-dimensional porous network structure with a porosity of 70-90% and an average pore size of 4.06 μm. The interface is clear and there is no phase separation phenomenon, providing an ideal carrier structure for the loading and release of drug molecules.

[0033] The preparation of the pesticide slow-release agent includes the following steps: (1) Weigh 80 mg of glycyrrhizic acid and 2 mg of azoxystrobin, dissolve them in 5 mL of methanol, and evaporate them at 37 °C to form a uniform film. (2) Add 1 mL of ultrapure water for hydration, then sonicate for 15 min (power 300 W) to disperse, then heat in a 40℃ water bath for 10 min and cool to room temperature. Repeat the heating-cooling process 3 times to finally form a transparent and elastic hydrogel.

[0034] 1. Morphological and performance characterization of pesticide slow-release agents: The microstructure was observed using scanning electron microscopy (SEM). The hydrogel samples were frozen in liquid nitrogen, freeze-dried for 48 hours, sputter-coated with gold, and then observed under a JSM-IT500 SEM. Figure 1 As shown.

[0035] The crystallization state of the pesticide slow-release agent (GA-AZOX) was analyzed by X-ray diffraction (XRD). A Bruker D8 diffractometer with a Cu Kα radiation source was used, and the scanning range was 5°–90°. Figure 2 As shown, pure azoxystrobin (AZOX) exhibits sharp crystallization diffraction peaks at 2θ = 18.88°, 26.47°, and 32.14°, while these characteristic peaks are significantly broadened and their intensity decreases in the co-assembled hydrogel, indicating that azoxystrobin is dispersed in an amorphous state in the supramolecular network constructed by glycyrrhizic acid (GA).

[0036] Intermolecular interactions were analyzed using Fourier transform infrared spectroscopy (FT-IR). An IRSpirit-X spectrometer was used with the KBr pellet method, and the scanning range was 500-4000 cm⁻¹. -1 The result is as follows Figure 3 As shown, the OH stretching vibration peak in the glycyrrhizic acid-pyraclostrobin gel is significantly broadened (approximately 3400 cm⁻¹). -1 This indicates enhanced hydrogen bonding; the CH vibration peak increased from 2935.1 cm⁻¹. -1 Displaced to 2978.5cm -1 This confirms the existence of hydrophobic interactions; the carbonyl peak (C=O, 1724 cm⁻¹) -1 The system remains stable. These results indicate that self-assembly is driven synergistically by hydrogen bonding and hydrophobic interactions.

[0037] The rheological properties were analyzed using a rotational rheometer (Kinexus Pro+). Frequency sweeps (0.1-10 Hz, 25℃) showed that the storage modulus (G′) was consistently higher than the loss modulus (G″), indicating that the system exhibits predominantly elastic response and stable solid-like behavior. Figure 4 Temperature scans (25-50℃) showed that the variations in G′ and G″ were less than 12.5%, and the tanδ value remained stable between 0.6 and 0.8, indicating good thermal stability. Figure 4 ).

[0038] The wettability was assessed using a contact angle meter (DSA25). The hydrogel was diluted 2-fold with ultrapure water, and 4 μL of the solution was dropped onto the surface of a tomato leaf for testing. The results showed that its contact angle was 80.78 ± 0.24°, significantly less than that of the azoxystrobin suspension (100.58 ± 0.46°). Figure 5This indicates that the gel has superior spreadability and adhesion, which is beneficial for leaf retention and drug efficacy.

[0039] 2. In vitro antibacterial test of pesticide slow-release agents: 2.1 Using Botrytis cinerea, the causal agent of tomato gray mold, as an indicator strain, an antibacterial experiment was conducted using the sample well method.

[0040] The pesticide slow-release agent was diluted with PBS buffer to a final concentration of 0, 10, 20, 30, and 50 μg / mL. 100 μL of each agent was added to the sample wells of a PDA plate, and a bacterial cake was inoculated in the center. The plates were then incubated in the dark at 25°C for 3-5 days. The colony diameter was measured and the inhibition rate was calculated.

[0041] The results are as follows Figure 6 As shown, the antibacterial effect exhibited a concentration-dependent pattern. The 50 μg / mL treatment group achieved an inhibition rate of 58.98% on day 5, significantly higher than the 10 μg / mL group (26.76%). The high-concentration groups (30-50 μg / mL) maintained a stable inhibition rate (53.91%-58.98%, RSD<2%) over 3-5 days, and dark brown deposits appeared at the colony edges, indicating that the drug exerted a long-lasting antibacterial effect by interfering with hyphal metabolism.

[0042] Further investigation was conducted into its pH-responsive release behavior. The hydrogel was placed in release media at pH 3 and pH 7, and 2.3 mL samples were taken on days 2, 6, 10, and 14, with each sample replaced with an equal volume of fresh media. The released solution was added to PDA medium containing bacteria, and the inhibition rate was calculated after incubation. The results are as follows: Figure 7 As shown, under pH=7 conditions, the antibacterial rate of the sampling group on day 10 was still 48.47% (RSD=0.49%) on day 5, and the release behavior was stable; while under pH=3 conditions, although the initial release was faster (the antibacterial rate was higher on day 2), it decreased significantly in the later stage, indicating that a neutral environment is more conducive to the continuous release and efficient utilization of drugs.

[0043] 2.2 Using *Early Blight of Tomato* and *Small Spot of Corn* as indicator strains, antibacterial experiments were conducted using the sample well method.

[0044] The pesticide slow-release agent was diluted with PBS buffer to a final concentration of 50 μg / mL for pyraclostrobin. 100 μL of the solution was added to the sample wells of a PDA plate, and the center of each plate was inoculated with *Early Blight of Tomato* and *Small Leaf Spot of Corn*. The plates were then incubated in the dark at 25°C for 5 days. The growth of the colonies was observed to evaluate the inhibitory effect of the glycyrrhizic acid-pyraclostrobin gel slow-release agent on these two plant pathogenic fungi.

[0045] The results are as follows Figure 8As shown, after 5 days of growth, the bacterial colonies treated with glycyrrhizic acid-pyraclostrobin gel were significantly smaller than those in the blank control group, especially against corn leaf blight pathogens, where glycyrrhizic acid-pyraclostrobin completely inhibited its growth. This fully demonstrates that glycyrrhizic acid-pyraclostrobin gel has a broad antibacterial spectrum.

[0046] 3. Verification of antibacterial effect through live inoculation experiment on tomatoes: Healthy tomato fruits were selected, and after surface disinfection, a 2×2 cm area was prepared on the peel, with a standard 0.5×0.5 cm wound made in the center. This area was then inoculated with *Botrytis cinerea* fungal cakes. Three treatment groups were set up: an experimental group (treated with a slow-release pesticide, pyraclostrobin at a final concentration of 62.5 μg / mL), a positive control group (glycyrrhizic acid hydrogel), and a blank control group (distilled water). Each group was replicated in 5 places and incubated at 25℃ for 1–4 days. The diameter of the lesions was measured daily, and the inhibition rate was calculated.

[0047] The results are as follows Figure 9 As shown, the experimental group achieved an inhibition rate of 95.4% on day 1, significantly higher than the control group. As time progressed, the inhibition rate of the experimental group remained at 21.85% by day 4, while the positive control group decreased to 5.04%, and the blank control group showed almost no antibacterial effect. No phytotoxicity was observed throughout the experiment, indicating that the gel system is safe for tomato fruits and possesses both high antibacterial efficiency and good biocompatibility.

[0048] 4. Biosafety Assessment – ​​Tomato Seed Germination Experiment: To comprehensively evaluate the biosafety of this slow-release agent, a tomato seed germination experiment was conducted. Three treatment groups were set up: the experimental group (pesticide slow-release agent, azoxystrobin concentration 0.002 mg / cm³) and the control group (pesticide slow-release agent, azoxystrobin concentration 0.002 mg / cm³). 2 The study included a positive control group (azoxystrobin-DMSO suspension, same concentration) and a blank control group (distilled water). Each group contained 50 seeds, which were disinfected and soaked in the solution for 6 hours. The seeds were then placed in a light incubator and cultured for 10 days. The number of germinations was recorded daily, and the root length, stem length, and fresh weight were measured on the 10th day.

[0049] The results are as follows Figure 10 As shown, the germination rate of the experimental group reached 23.33% on day 4, higher than that of the blank control group (13.33%) and the positive control group (20%). On day 7, the germination rate of both the experimental and positive control groups reached 100%, but the experimental group exhibited a higher uniformity of germination (95%), with well-developed root systems, a taproot length of 8.50 cm, and a significantly higher fresh weight than the control group. Some seedlings in the positive control group showed cotyledon yellowing, presumably due to the toxicity of DMSO solvent. The experimental group, however, completely avoided the use of organic solvents, indicating superior biocompatibility and application potential.

[0050] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A pesticide slow-release agent, characterized in that, Hydrogels with a three-dimensional porous network structure are formed by the self-assembly of glycyrrhizic acid or glycyrrhetinic acid and pyraclostrobin through intermolecular non-covalent interactions. The porosity is 70%-90% and the average pore size is 2-5μm.

2. The pesticide slow-release agent according to claim 1, characterized in that, The mass ratio of glycyrrhizic acid or glycyrrhetinic acid to pyraclostrobin is 30-80:1-6, preferably 40:

1.

3. A method for preparing a pesticide slow-release agent, characterized in that, Includes the following steps: (1) Glycyrrhizic acid or glycyrrhetinic acid and pyraclostrobin are dissolved in an organic solvent and then evaporated by rotary evaporation to form a film; (2) Add hydration medium, disperse by ultrasonication, and form a stable hydrogel by cyclic heating-cooling.

4. The method for preparing the pesticide slow-release agent according to claim 3, characterized in that, In step (1), the organic solvent is selected from one or more of methanol, ethanol, and acetonitrile, with methanol being preferred.

5. The method for preparing the pesticide slow-release agent according to claim 3, characterized in that, In step (2), the hydration medium is selected from one or more of water and phosphate buffer, preferably water.

6. The method for preparing the pesticide slow-release agent according to claim 4 or 5, characterized in that, The amount of glycyrrhizic acid or glycyrrhetinic acid dissolved in each 10 ml of the organic solvent is 60-160 mg; the volume ratio of the hydration medium to the organic solvent is 1:(2-6), preferably 1:

5.

7. The pesticide slow-release agent according to claim 3, characterized in that, In step (2), the power of the ultrasound is 50-200W and the duration of the ultrasound is 5-15min.

8. The pesticide slow-release agent according to claim 3, characterized in that, In step (2), the number of cycles is 1-5, the heating temperature is 40-60℃, the heating time is 5-30 min, and the cooling temperature is room temperature.

9. The application of the pesticide slow-release agent as described in claim 1 or 2, or the pesticide slow-release agent prepared by the preparation method of any one of claims 3-8, in inhibiting agricultural pathogens.

10. The application according to claim 9, characterized in that, The pathogens are tomato gray mold, tomato early blight, corn leaf spot, wheat powdery mildew, and grape downy mildew.

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