CN115702640A - A pesticide sustained-release preparation with induced resistance and ROS response, its preparation method and application - Google Patents

Abstract

The invention discloses a pesticide slow-release preparation with induction resistance and ROS response, a preparation method and application thereof, and belongs to the technical field of pesticides. Pesticide sustained-release preparations are prepared by loading pesticides on polymer materials with induced resistance and ROS response. Between 15‑75%. The carrier material of the pesticide slow-release preparation with induced resistance and ROS response itself has induced resistance. The pesticide slow-release preparation of the invention can reduce the diffusion, digestion and loss of the pesticide in the environment, prolong the effective period of the pesticide, and reduce the frequency of spraying, thereby reducing the dosage of the pesticide and protecting the environment. And at the same time, it shows the characteristics of ROS responsiveness, induction of plant resistance and promotion of plant growth.

Description

A pesticide sustained-release preparation with induced resistance and ROS response and preparation method thereof and application

technical field

The invention belongs to the technical field of pesticides, and in particular relates to the preparation and biological effects of a pesticide slow-release preparation with resistance and ROS response.

Background technique

Pesticides have the advantages of high efficiency, quickness, economy, simple operation, and low labor intensity in the control of crop diseases, insect pests and weeds, and play an important role in the integrated pest control. China has a huge production and usage of chemical pesticides, mainly conventional pesticide formulations such as emulsifiable concentrates, wettable powders and water-emulsions. However, conventional pesticide formulations have problems such as large amount of organic solvents used, dust drift, poor dispersibility, etc., and do not have the ability to control the release of active ingredients, resulting in the loss of more than 90% of the active ingredients of pesticides in the environment near the target. The long-term large-scale and low-efficiency application of pesticides has caused the pesticide residues in vegetables, fruits, grains, soil and water to exceed the standard in my country, which not only poses a serious threat to non-target organisms and human health, but also leads to the destruction of the structure and function of the ecosystem. .

In recent years, due to the advantages of targeted release, controllable release, and long-lasting effect, pesticide slow- and controlled-release formulations have become a hot spot in the research of pesticide formulations. Controlled release technology means that the active ingredients in a specific system can be continuously released to a specific target according to the designed concentration and time, and achieve the expected effect.

Currently, although environmental response systems are well developed in medicine, their application in agriculture is still limited. Although simple sustained release has been achieved, responsive release materials based on environmental changes have not yet been developed. The field of pesticides also needs to continue to systematically research, develop and improve environmentally responsive, targeted, and controlled-release pesticides. In particular, efficient systems that respond to internal biological stimuli represent a great challenge in this field.

ROS is a general term for a class of oxygen-containing substances with active chemical properties and strong oxidizing ability, including O ₂ ^- , OH, O ₂ , H ₂ O ₂ and lipid peroxidation products (lipoxyl radical LO, lipid peroxidation material LOO, etc.). Under normal growth conditions, the antioxidant system in plants can keep the production and removal of ROS in a dynamic balance; however, under biotic and abiotic stress, the antioxidant mechanism in plants is destroyed, and the balance of ROS production and removal It is broken, resulting in the excessive accumulation of ROS and the phenomenon of oxidative burst, which is also recognized as one of the plant's disease resistance responses. During the interaction between many pathogens and plants, reactive oxygen species in plants participate. Plant defense responses induced by pathogens are often accompanied by a burst of reactive oxygen species.

Contents of the invention

Based on the above background technology, the primary purpose of the present invention is to prepare a pesticide sustained-release preparation with induced resistance and ROS response, its preparation method and application.

The object of the present invention is achieved through the following technical solutions:

One aspect of the present invention provides a pesticide slow-release formulation with induced resistance and ROS response, the pesticide sustained-release formulation includes a carrier with induced resistance and ROS response and loaded pesticides, and the pesticide with induced resistance and ROS response The structural formula of the carrier is as follows:

R ₁ -RR ₂

R is a ROS response group, R ₁ is a segment with induction resistance, and R ₂ is a carrier matrix segment;

ROS responsive groups include thioethers, thioketals, selenides, diselenides, aromatic borates, oxalates, ferrocenes; segments with inductive resistance include chitooligosaccharides, chitosan, seaweed polysaccharides . Oxypolyethylene glycol, polycaprolactone, 2-mercaptoimidazoline, cholesterol.

Based on the above technical scheme, preferably, the pesticide is glyphosate, Jinggangmycin, propamocarb, propamocarb hydrochloride, ethephon, trichlorfon, chlordimeform, dimehypo, dimethyltetrachlor , Dithionate.

Based on the above technical solution, preferably, the pesticide loading amount of the pesticide sustained-release preparation accounts for 10-90% by weight of the carrier material, and the particle size of the pesticide sustained-release preparation is between 10-800nm.

Based on the above technical solution, preferably, the pesticide sustained-release preparation can induce plant resistance.

Based on the above technical scheme, preferably, the pesticide sustained-release preparation is released under the condition of aqueous solution, releasing 5-10% within 24h, releasing 40-50% within 480h; in the ROS solution, releasing 10-20% within 24h, releasing within 480h 95-99% released within.

Based on the above technical solution, preferably, the ROS solution is a solution containing at least one of O ₂ ^- , HO ₂ ^- , and -OH at a concentration of 4 μM-1M, preferably hydrogen peroxide, potassium peroxide, calcium peroxide, peroxide barium oxide solution.

Another aspect of the present invention provides a preparation method of the above-mentioned pesticide sustained-release preparation, the preparation method comprising the following steps:

S1: Preparation of a carrier with inducible resistance and ROS response: The segment with inducible resistance is sequentially connected to the ROS responsive group and the carrier matrix segment through covalent bonds to obtain a polymer material with inducible resistance and ROS response ;

S2: Preparation of pesticide-loaded polymer micelles: dissolve the polymer material with induced resistance and ROS response prepared in S1 and pesticides in a solvent to obtain an oil phase, drop the oil phase into the water phase, stir, and dialyze Unloaded pesticides are removed, and the pesticide sustained-release preparation is obtained by drying after dialysis.

Based on the above technical scheme, preferably, the mass ratio of the polymer material with induced resistance and ROS response to the pesticide is 2:1-5:1.

Based on the above technical solution, preferably, the volume ratio of the oil phase to the water phase is 1:10-1:1, the stirring time is 2-24 hours, and the dialysis time is 12-48 hours.

Based on the above technical solution, preferably, the solvent includes methanol, acetonitrile, dichloromethane, and DMSO.

Based on the above technical solution, preferably, the water phase includes water, PBS solution, PVA solution or sodium chloride solution; the concentration of the PBS solution, PVA solution or sodium chloride solution is 0.01-10M.

Another aspect of the present invention provides a pesticide composition comprising the above-mentioned sustained-release pesticide formulation.

Another aspect of the present invention provides an application of the above pesticide sustained-release preparation, which is used for the prevention and control of crop diseases and insect pests.

Based on the above technical scheme, preferably, the dosage is 1/10-1/50 of the conventional dosage.

Beneficial effect

(1) The present invention uses phytopathological phenomena to creatively load pesticides such as Jinggangmycin and glyphosate with antibacterial effects in materials with oxidation responsiveness, which can reduce the diffusion, digestion and loss of pesticides in the environment, and at the same time Extend the duration of pesticides, reduce the frequency of application, reduce the amount of pesticides and protect the environment.

(2) The creative invention of the present invention enables both water-soluble and oil-soluble pesticides to be loaded and realize responsive slow release.

(3) The pesticide slow-release agent of the present invention can spontaneously release pesticides according to the change of ROS content in the natural process of pathogenic bacteria infecting plants, accurately grasp the timing of drug release, increase the targeting of drugs, release time is long, and drug efficacy can be maintained For more than 20 days, the frequency of re-spraying of pesticides is reduced, and the amount of pesticides used is reduced. The dosage is 1/10-1/50 of the conventional dosage.

(4) The pesticide slow-release agent of the present invention, the oligosaccharide contained in the carrier material can induce plant resistance, act as a plant growth regulator, promote cell activation, and stimulate growth.

(5) The pesticide slow-release agent of the present invention has a simple and feasible preparation process. Compared with conventional commercially available pesticides, it reduces the use of organic solvents, without additives such as stabilizers, and the loaded material itself has biocompatibility and biodegradability It is non-toxic and will not cause any pressure on the environment, and its antibacterial effect is 10-50 times better than that of commercially available agents.

Description of drawings

FIG. 1 is the mPEG-TK ¹ H-NMR spectrum in Example 1.

FIG. 2 is the mPEG-TK-COS ¹ H-NMR spectrum in Example 1.

Fig. 3 is a transmission electron microscope image of Vail@mPEG-TK-COS prepared in Example 1.

FIG. 4 is the ¹ H-NMR spectrum of mPEG-TK-COS in Example 3 after ROS response cleavage.

Fig. 5 is the in vitro release diagram of Vail@mPEG-TK-COS with induction resistance and ROS response in Example 4.

Figure 6 is the antibacterial effect of the Vail@mPEG-TK-COS plate with induced resistance and ROS response in Example 5. Figure A is the plate antibacterial picture, and B is the statistics of the colony diameter of Rhizoctonia solani.

Figure 7 is the antibacterial effect of Vail@mPEG-TK-COS with induced resistance and ROS response in Example 6, A is the H ₂ O treatment group, B is the Vail treatment group, and C is the mPEG-TK-COS treatment group , D is the Vail@mPEG-TK-COS treatment group, E is the lesion ratio statistics.

Fig. 8 is the disease index statistics of Vail@mPEG-TK-COS drug efficacy with induced resistance and ROS response in Example 7.

Fig. 9 is the test result of rice hormone in Example 7.

Detailed ways

The present invention will be further described below in conjunction with specific examples. The raw materials of the present invention are commercially available unless otherwise specified.

Example 1

S1: Preparation of sustained-release formulations of pesticides with induced resistance and ROS response: (1) Synthetic thioketal (TK): 5.2 g of 3-mercaptopropionic acid and 5.8 g of anhydrous acetone, dissolved in dry hydrogen chloride, at room temperature Stir for 6h. The polymer after stirring was placed on ice until the crystallization was complete, dried in vacuo with hexane and cold water to obtain a white product, dried to obtain a constant weight product, and set aside.

(2) Synthesis of mPEG-TK: 1.01g TK, 1.54g carbodiimide, 0.92g N-hydroxysuccinimide, 1.03g N,N-diisopropylethylamine, dissolved in 50mL dichloromethane, 600rpm After stirring at room temperature for 2 hours, 4.0 g of mPEG was added, the solid dissolved and became a clear solution, stirred at room temperature at 600 rpm for 48 hours, washed with ether three times, dried to obtain a white powder, and set aside. ^{The 1} H-NMR spectrum is shown in Figure 1. It can be seen from the figure that 1.60, 2.60-2.68, 2.86-2.90ppm are the characteristic absorption peaks of ketal thioketal, which proves the successful preparation of mPEG-TK.

(3) Synthesis of mPEG-TK-COS: 2.23g mPEG-TK, 0.29g carbodiimide, 0.78g, N,N-diisopropylethylamine, dissolved in 10mL DMSO, stirred at room temperature at 600rpm for 2h , add 2.4g chitosan oligosaccharide (COS), after the solid dissolves, it changes from a clear solution to a brown solution, stir at 600rpm in the dark for 48h at room temperature, wash with ether three times, dry to obtain a powder, and set aside, ^{the 1} H-NMR spectrum is as follows: As shown in Figure 2, it can be seen from the figure that 4.51-4.61ppm is the characteristic absorption peak of COS, which proves the successful preparation of mPEG-TK-COS.

S2: Preparation of the sustained-release formulation Vail@mPEG-TK-COS loaded with Jinggangmycin (Vail): 1g mPEG-TK-COS and 0.5g Vail were simultaneously dissolved in 5mL DMSO and ultrasonically oscillated for 30min to completely dissolve and mix evenly as oil Phase, use a syringe pump to add drop by drop into 50mL water at a constant speed. After the drop is completed, stir at 600rpm at room temperature overnight to form a carrier material, put it into a dialysis bag with a molecular weight cut-off of 500-1000, and dialyze with pure water for 48h. Change the water every 2 hours until there is no absorption value at 210nm, dry it for later use, and detect it with a transmission electron microscope. The morphology of Vail@mPEG-TK-COS is shown in Figure 3. The successful preparation of drug-loaded sustained-release preparations was monodisperse spherical with uniform size.

Example 2

S1: Preparation of pesticide sustained-release formulations with induced resistance and ROS response: (1) synthetic cholesterol-oxalate: 1.01g oxalate, 1.54g carbodiimide, 0.92g N-hydroxysuccinimide, 1.03g of N,N-diisopropylethylamine, dissolved in 50mL of dichloromethane, stirred at room temperature at 600rpm for 2h, then added 4.0g of cholesterol, the solid dissolved and became a clear solution, stirred at room temperature at 600rpm for 48h , washed three times with ether, dried to give a white powder, and set aside.

(2) Synthesis of cholesterol-oxalate-pectin oligosaccharides: In an ice-water bath under a nitrogen atmosphere, 0.23g of cholesterol-oxalate, 0.012g of 4-dimethylaminopyridine and 0.412g of N,N'-bis Cyclohexylcarbodiimide was dissolved in 50 mL of dichloromethane. Then, a DMSO solution in which 0.5 g of pectin oligosaccharide was dissolved was added dropwise. The reaction mixture was stirred at room temperature for 48 h, then filtered to remove a white precipitate. After washing, the resulting precipitate was the product. The product cholesterol-oxalate-pectic oligosaccharide was vacuum-dried at 25° C. for 6 hours, and set aside.

S2: Preparation of glyphosate-loaded sustained-release preparation: 2.5g of cholesterol-oxalate-pectic oligosaccharide and 0.5g of glyphosate were simultaneously dissolved in 100mL DMSO and ultrasonically oscillated for 30min to completely dissolve and mix uniformly as the oil phase. Use a syringe pump to add drop by drop into 10mL water at a constant speed. After the drop is completed, stir at 600rpm at room temperature overnight to form a carrier material, put it into a dialysis bag with a molecular weight cut-off of 500-1000, and dialyze with pure water for 48 hours, every 2 hours Change the water once until there is no absorption value at 210nm, and dry it for later use.

Example 3

Verification of ROS-responsive properties of carrier material mPEG-TK-COS: preparation of H ₂ O ₂ mimics ROS stimulation. Add 0.02 g of carrier material to a centrifuge tube containing 5 mL of 10 mM H ₂ O ₂ , incubate for 48 h and freeze-dry, then use 400 M NMR to characterize its structure. As shown in Figure 4, the vibration absorption peak at 1.60 disappears, indicating that there is ROS The responsive group was broken, which proved its ROS responsiveness.

Example 4

In vitro release test of Vail@mPEG-TK-COS loaded with Jinggangmycin, which has induced resistance and ROS response: Weigh a certain amount of dried Vail@mPEG-TK-COS prepared in Example 2 into a dialysis bag (molecular weight cut-off 500-1000), respectively placed in water and 80 μM hydrogen peroxide buffer solution, respectively, at intervals of 1h, 2h, 4h, 8h, 12h, 24h, 48h to 20 days. Samples were taken for testing and rehydration. The sample was detected by high performance liquid chromatography at 210nm, and the release curve was drawn as shown in Figure 5. It can be seen from the figure that Vail@mPEG-TK-COS released quickly under the action of H ₂ O ₂ , without H The sustained-release preparations in the presence of ₂ O ₂ released a lower amount.

Example 5

Antibacterial activity of Vail@mPEG-TK-COS with induced resistance and ROS response: In this experiment, the bactericidal efficacy of pesticide sustained-release formulations against Rhizoctonia solani was determined by the growth rate method. On the basis of the preparation experiment, Jinggangmycin with an effective concentration of 1.57-25 mg.L ^-1 and Vail@mPEG-TK-COS prepared in Example 2 were used respectively, so that the pesticide sustained-release preparation was set at 10-90% inhibition Within the range of the rate, after resurrection and re-identification, the fungal cake of Rhizoctonia solani (4mm in diameter) was inoculated on a petri dish containing PDA medium, and cultivated in a constant temperature incubator at 28°C. Determine the relative inhibition percentage of pesticide sustained-release preparations to fungi by comparing the colony diameter, as shown in Figure 6, compared with the control group and commercially available Vail, 1.56mg.L ^-1 , 6.75mg.L ^-1 , 25mg.L ^- The antibacterial effect of Vail@mPEG-TK-COS of ¹ is more obvious, and the antibacterial effect increases with the increase of the concentration. itself has antibacterial activity. Due to the synergistic effect of the bacteriostatic effect of the material itself and the drug, the pesticide sustained-release preparation with the ability to induce plant resistance and ROS response is obviously better than the pesticide alone. The above experiments prove that the effect of the drug-loaded sustained-release preparation prepared by us is better than that of the commercially available drugs.

Example 6

In vitro antibacterial effect of Vail@mPEG-TK-COS with ROS response and induction of plant resistance: In this experiment, the bactericidal efficacy of pesticide sustained-release formulations against Rhizoctonia solani was determined by the growth rate method. On the basis of the preparation experiment, the method for inoculating the isolated leaves with Rhizoctonia solani is as follows: cut the length of rice with the same growth time and width into 4-5 cm, place it in a place containing benzimidazole (0.1gL ^-1 ) Petri dish on water agar medium. The anterior surface of the leaf (the side without veins) is in close contact with the medium. Spray Vali, Vail@mPEG-TK-COS, mPEG-TK-COS and ddH ₂ O on detached leaves (the effective concentration of Vali is 25 mg.L ^-1 ). PDA agar blocks bearing R. solani hyphae with a diameter of 5 mm were removed at the same distance from the sclerotia with a sterile punch. The above-mentioned agar block with hyphae is placed in the center of the vein of the isolated rice leaf. The culture dish was cultured in a constant temperature incubator at 28°C. By calculating the ratio of the diseased leaf spot area to the entire leaf area, the disease of the fallen leaves was calculated. The formula is as follows.

S1/S2=P1/P2

S1: the area of the diseased spot on the detached rice leaf; S2: the area of the detached rice leaf; P1: the pixel of the diseased spot; P2: the pixel of the rice

As shown in Figure 7, compared with the control group and Vail, the antibacterial effect of the drug-loaded sustained-release preparation was more obvious, and the proportions of lesion area in the water, Vail, empty carrier material and Vail@mPEG-TK-COS treatment groups were respectively 50.56%, 20.07%, 18.26% and 5.6%, similar to the results of the plate antibacterial test, the carrier material with induced resistance has a significant difference in antibacterial effect compared with the control group. Vail@mPEG-TK-COS, which has the ability to induce resistance and ROS response at the same time, is significantly better than the single pesticide due to the synergistic effect of the carrier material inducing the resistance of the plant itself and the drug. The above experiments prove that the drug-loaded sustained-release preparation prepared by us has a very significant difference compared with the effect of Jinggangmycin, and the effect is better.

Example 7

Germinated rice (Oryza sativa spp. japonica cv. Nipponbare) seedlings were sown into the Yoshida solution. The cultivation conditions of the rice plants were 80-100% relative humidity, 14 hours of light at 28°C and 10 hours of darkness at 24°C. Rice cultured for 30 days was inoculated with Rhizoctonia solani using the "toothpick insertion method". The specific method is as follows: Cut a commercially available toothpick into small sections of about 8 mm, and then divide each section into 4 parts evenly. Place the treated toothpicks neatly on the bottom of the Petri dish. Then add potato dextrose medium on a toothpick. Take a 5 mm diameter piece of PDA agar at the same distance from the sclerotia with a sterile puncher and place it at five points at the same distance. Place the Petri dish in an incubator at 28 °C until the fungi are filled with toothpicks. The rice was sprayed with Vail and Vail@mPEG-TK-COS prepared in Example 1 (the effective concentration of Vail was 25 mg.L ^-1 ). Take the toothpick segment containing the fungus with sterile tweezers and insert it into the sheath of the penultimate leaf of rice. There were 30 rice plants per treatment, and three replicates were performed for each treatment group. The statistical method of the disease index is as follows:

And extract the hormone detection of rice disease spots.

As shown in Figure 8, compared with the control group and Vail, the antibacterial effect of Vail@mPEG-TK-COS is more obvious. After the prepared Vail@mPEG-TK-COS was treated, the content of salicylic acid (SA) in rice lesions increased significantly, which proved that it induced resistance in rice plants. The above experiments proved that the pesticide-loaded slow-release formulation we prepared had The characteristics of inducing plant resistance and ROS response, its efficacy is significantly better than that of a single pesticide.

Claims (10)

1. The pesticide slow-release preparation with the functions of inducing resistance and ROS response is characterized by comprising a carrier with the functions of inducing resistance and ROS response and a loaded pesticide, wherein the structural formula of the carrier with the functions of inducing resistance and ROS response is as follows:

R ₁ -R-R ₂

r is an ROS-responsive group, R ₁ Being a segment with induced resistance, R ₂ Is a carrier matrix segment;

ROS-responsive groups include thioethers, thioketals, selenides, diselenides, aromatic borates, oxalates, ferrocenes; the chain segment with induction resistance comprises chitosan oligosaccharide, chitosan, algal polysaccharide, chitooligosaccharide, chitin, pectin oligosaccharide, exopolysaccharide, pectin, galacturonic acid, glucooligosaccharide and glucan, and the carrier matrix chain segment comprises polyethylene glycol, methoxy polyethylene glycol, polycaprolactone, 2-mercaptoimidazoline and cholesterol.

2. The sustained-release pesticide formulation according to claim 1, wherein the pesticide is glyphosate, validamycin, propamocarb hydrochloride, ethephon, trichlorfon, chlordimeform, dimehypo, methoxone, and ethephon.

3. The pesticide sustained-release preparation according to claim 1, wherein the pesticide loading amount of the pesticide sustained-release preparation is 10-90% by weight of the carrier material, and the particle size of the pesticide sustained-release preparation is 10-800 nm.

4. The pesticide sustained-release preparation according to claim 1, wherein the pesticide sustained-release preparation is released under the condition of aqueous solution, and is released within 24h by 5-10% and within 480h by 40-50%; in ROS solution, 10-20% is released within 24h, and 95-99% is released within 480 h.

5. The method for producing a sustained-release preparation of agricultural chemicals according to any one of claims 1 to 4, characterized by comprising the steps of:

s1: preparation of vectors with induced resistance and ROS response: connecting the chain segment with induced resistance with an ROS response group and a carrier matrix chain segment in sequence through a covalent bond to obtain the polymer material with induced resistance and ROS response;

s2: preparing pesticide-loaded polymer micelles: and (2) dissolving the polymer material with induced resistance and ROS response prepared in the step (S1) and the pesticide in a solvent to obtain an oil phase, dripping the oil phase into a water phase, stirring, dialyzing to remove the unloaded pesticide, and drying after dialysis to obtain the pesticide slow-release preparation.

6. The preparation method according to claim 5, wherein the mass ratio of the polymeric material having induced resistance and ROS response to the pesticide is 2.

7. The preparation method according to claim 5, wherein the volume ratio of the oil phase to the water phase is 1.

8. The method of claim 5, wherein the solvent comprises methanol, acetonitrile, dichloromethane, DMSO.

9. The method for preparing according to claim 5, wherein the aqueous phase comprises water, a PBS solution, a PVA solution or a sodium chloride solution; the concentration of the PBS solution, the PVA solution or the sodium chloride solution is 0.01-10M.

10. The use of a sustained-release pesticide formulation according to any one of claims 1 to 4 for the control of crop pests.

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