CN114668016B - Zein loaded emamectin benzoate nanometer slow-release agent and preparation method and application thereof - Google Patents
Zein loaded emamectin benzoate nanometer slow-release agent and preparation method and application thereof Download PDFInfo
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/08—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing solids as carriers or diluents
- A01N25/10—Macromolecular compounds
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/24—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing ingredients to enhance the sticking of the active ingredients
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Dentistry (AREA)
- Plant Pathology (AREA)
- Engineering & Computer Science (AREA)
- Pest Control & Pesticides (AREA)
- Agronomy & Crop Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Toxicology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention provides a zein loaded emamectin benzoate nanometer slow release agent, a preparation method thereof and application thereof as an insecticide for preventing and controlling lepidoptera insects, the zein loaded emamectin benzoate nanometer slow release agent is prepared from zein, emamectin benzoate, ethanol and water, and can carry out emamectin benzoate response type release along with the change of pH and/or enzyme in the environment. The zein-loaded emamectin benzoate nanometer slow-release agent provided by the invention has the advantages of smaller particle size, good particle dispersibility, better wettability and slow-release performance, improved effective utilization rate of pesticides and obviously improved prevention and control effect on lepidopteran insects.
Description
Technical Field
The invention belongs to the technical field of pesticides. In particular to a zein loaded emamectin benzoate nanometer slow release agent, a preparation method and application thereof.
Background
The traditional pesticides have the problems of large using amount of organic solvents and poor dispersibility, and are mostly open systems, so that most of the pesticides are lost in the environment. Aiming at the problem, the precise controlled release technology is a new direction of research. The accurate controlled release technology takes the physicochemical property of pesticides as a research basis, selects proper carrier materials and administration ways, appropriately serves farmlands in good time, and carries out accurate controlled release on pest targets, thereby reducing the effective demands of crops on pesticides to the minimum, and gradually achieving the purposes of drug utilization, safety, adjustability, intellectualization and the like from direct release to slow release to controlled release. However, the current research on pesticide controlled release can only simply control the release rate of the pesticide, and can not enable the pesticide to be released in response to the environmental requirements, so that the usage amount of the pesticide is increased, and the effective utilization rate of the pesticide is reduced.
Insects are the most numerous animal groups on the earth, and are various in variety, different in form and very wide in range of motion; the lepidoptera insects are used as the second largest of the class of insects, the quantity of the lepidoptera insects accounts for about 16 percent of the total quantity of insects, and the insects in agriculture and forestry are more in species, so that the insect population has great threat to agricultural production. The study shows that the digestive tract structure of lepidopteran insect larvae is quite simple and is mainly divided into 3 parts of the foregut, the midgut and the hindgut (see figure 1), the intestinal environment is generally alkaline, the midgut is most alkaline in general, and the foregut and the hindgut are weaker in alkalinity. Therefore, if the drug can be targeted released aiming at the alkaline intestinal canal of lepidopteran insects, an environment response type targeting accurate release system is designed, so that the drug is kept in a relatively stable state in a neutral environment, but the release speed is increased in the alkaline intestinal canal environment, the drift of pesticides can be reduced, the action rate of the pesticides on the targets is improved, and the method has a great development prospect in reducing pesticide residues and environmental pollution.
Therefore, there is a need in the art to develop a pesticide microcapsule capable of being released in response to changes in environmental factors such as pH, enzymes, etc., so as to effectively kill lepidopteran insects and increase the rate of action of the pesticide on the target.
Disclosure of Invention
Aiming at the problems, the invention aims to provide the zein-loaded emamectin benzoate nanometer slow-release agent which can be released in response to the changes of pH and enzyme in the environment, so that lepidopteran insect larvae are effectively killed, the acting rate of pesticides on targets is obviously improved, pesticide residues and environmental pollution are reduced, and the preparation method is simple, easy to operate and good in repeatability.
The invention also aims to provide a preparation method of the zein-loaded emamectin benzoate nanometer slow-release agent.
The invention also aims to provide the application of the zein-loaded emamectin benzoate nanometer slow release agent as an insecticide for preventing and controlling lepidoptera insects, preferably asparagus caterpillar.
In order to achieve the aim, the invention provides a zein-loaded emamectin benzoate nanometer slow-release agent which is prepared from zein, emamectin benzoate, ethanol and water, wherein the zein-loaded emamectin benzoate nanometer slow-release agent can carry out emamectin benzoate response type release along with the change of pH and/or enzyme in the environment.
In some embodiments, the zein-loaded emamectin benzoate nano-release agent has a higher emamectin benzoate release rate in a pH alkaline environment than in a pH neutral environment;
Preferably, the zein-loaded emamectin benzoate nano slow release agent can carry out emamectin benzoate response type release along with the change of alkaline protease in the environment;
Preferably, the zein-loaded emamectin benzoate nano-delivery vehicle has an increased emamectin benzoate release rate in the presence of alkaline protease compared to the absence of alkaline protease.
In some embodiments, the zein-loaded emamectin benzoate nano slow release agent has an encapsulation rate of 33.3-50.8%, preferably 45.6%, and a drug loading rate of 7-21.3%, preferably 21.3%;
preferably, the particle size of the zein loaded emamectin benzoate nanometer slow release agent is 100-200nm.
The invention further provides a preparation method of the zein-loaded emamectin benzoate nanometer slow release agent, which comprises the following steps of:
Step a: dissolving zein and emamectin benzoate in an ethanol aqueous solution to obtain an organic solution;
Step b: c, injecting the organic solution obtained in the step a into water to volatilize the organic solvent, so as to obtain the zein-loaded emamectin benzoate nanometer slow release agent.
In some embodiments, the method of making comprises the steps of:
Step a: dissolving zein and emamectin benzoate in ethanol water solution, and carrying out vortex oscillation at the speed of 300r/min for 2-4min to obtain an organic solution;
step b: c, injecting the organic solution obtained in the step a into water at 37 ℃, stirring for 1-3 hours, preferably 3 hours, at a constant temperature and 300r/min, and volatilizing the organic solvent to obtain the zein-loaded emamectin benzoate nanometer slow release agent.
In some embodiments, the volume fraction of ethanol in the aqueous ethanol solution in step a is 70%;
preferably, the organic solution in step a is a yellow clear transparent organic solution.
In some embodiments, the concentration of zein in the organic solution obtained in step a is between 5 and 15g/L, preferably 5g/L;
Preferably, the concentration of emamectin benzoate in the organic solution obtained in step a is 2.5-10g/L, preferably 5g/L;
Preferably, the weight ratio of emamectin benzoate to zein in the organic solution obtained in the step a is 1:6-2:1, preferably 1:1.
In some embodiments, in step b, the volume ratio of organic solution to water is 1:20;
preferably, in the step b, the organic solution obtained in the step a is injected into water by a pipette.
The invention further provides application of the zein-loaded emamectin benzoate nanometer slow-release agent as an insecticide for preventing and controlling lepidoptera insects, preferably spodoptera exigua.
Zein (Zein) is selected as a carrier material in the present invention. Zein is natural protein with good biocompatibility, biodegradability and easy film formation in nature, raw materials are cheap and easy to obtain, degradation products are amino acid and polypeptide, and environmental pollution is avoided. In addition, zein has the advantages of moisture resistance, ultraviolet resistance and adhesiveness, and can enable the residence time of the medicine on the surface of a target to be longer, thereby being beneficial to the absorption and conduction of the medicine, improving the stability of the medicine, and further greatly improving the effective utilization rate of the medicine.
Zein is stable in gastric acid but is degraded in the insect gut by alkaline proteases therein, which makes it useful for preparing drug delivery systems that respond to the insect's alkaline gut. In addition, the zein has good solvent self-assembly sphericity, so that the zein can be synthesized into nano particles without depending on an emulsifying agent, and can be prepared by using a phase separation method.
The invention selects emamectin benzoate (collectively referred to as emamectin benzoate and EMB) as the pesticide active ingredient to prepare the nano pesticide slow-release agent. The emamectin benzoate is a high-efficiency, broad-spectrum, low-toxicity and pollution-free biological source pesticide, has higher insecticidal activity, has extremely high biological activity on lepidoptera pests such as plutella xylostella, mites and the like, and is safe and low-toxic for human beings and mammals. However, the emamectin benzoate has a macrolide structure, belongs to avermectin compounds, and has the defects of short duration, easiness in photolysis and easiness in adsorption by soil, so that the application and performance of the emamectin benzoate are limited. According to the invention, zein is selected as a carrier material to load emamectin benzoate, and the nano pesticide slow release agent is prepared by optimizing the preparation process and parameters, so that the duration of the emamectin benzoate can be remarkably prolonged, the defects of easiness in photolysis and adsorption by soil are overcome, and the effective pesticide utilization rate is improved.
The inventor finds through multiple experiments that in the preparation method of the zein-loaded emamectin benzoate nanometer slow-release agent, the stirring time in the step b and the concentration of zein and the concentration of emamectin benzoate in the organic solution in the step a have influence on the particle size of the zein-loaded emamectin benzoate nanometer slow-release agent, wherein the influence of the stirring time on the particle size is the greatest, the influence of the concentration of zein on the particle size is the least, and the influence of the concentration of emamectin benzoate on the particle size is the least.
The inventor finds through multiple experiments that in the preparation method of the zein-loaded emamectin benzoate nanometer slow-release agent, the concentration of the emamectin benzoate in the organic solution in the step a, the concentration of the zein and the stirring time in the step b all have influences on the encapsulation efficiency of the zein-loaded emamectin benzoate nanometer slow-release agent, wherein the concentration of the emamectin benzoate in the step a has the largest influence on the encapsulation efficiency, the concentration of the zein has the secondary influence on the encapsulation efficiency, and the stirring time has the smallest influence on the encapsulation efficiency.
The inventor finds through multiple experiments that in the preparation method of the zein-loaded emamectin benzoate nanometer slow-release agent, the concentration of the emamectin benzoate in the organic solution in the step a, the concentration of the zein and the stirring time in the step b have influence on the drug loading rate of the zein-loaded emamectin benzoate nanometer slow-release agent, wherein the influence of the concentration of the emamectin benzoate in the step a on the drug loading rate is the largest, the influence of the concentration of the zein on the drug loading rate is the second, and the influence of the stirring time on the drug loading rate is the smallest.
The inventor comprehensively considers the influences of the proportion of each component and the technological parameters in the preparation method of the zein-loaded emamectin benzoate nanometer slow-release agent on the factors such as the pesticide particle size, the encapsulation efficiency, the drug loading rate, the slow-release performance, the insecticidal effect and the like, and obtains the optimal proportion of the components and the technological parameters.
The zein-loaded emamectin benzoate nanometer slow-release agent prepared by adopting the zein as a carrier material and emamectin benzoate as a pesticide active ingredient and adopting a phase separation method has the following advantages compared with the prior art:
1. The zein-loaded emamectin benzoate nanometer slow release agent prepared by the invention has smaller particle size (the nanometer level can reach about 100-200 nm), good particle dispersibility and uniform distribution, improves the contact area of pesticides and plant leaf surfaces, increases the adhesiveness and permeability of the pesticides on the plant leaf surfaces, reduces pesticide loss, improves the effective utilization rate of the pesticides, and obviously improves the prevention and treatment effect on lepidopteran insects;
2. the zein-loaded emamectin benzoate nano slow release agent can be stored at different temperatures, can keep stable and uniform dispersion, has no obvious change in particle size, and has good storage stability;
3. The zein-loaded emamectin benzoate nanometer slow-release agent can effectively reduce the photodegradation rate of active ingredient emamectin benzoate, obviously improve the photostability of the emamectin benzoate under the field illumination condition, greatly reduce the decomposition loss of the emamectin benzoate and effectively prolong the drug effect time;
4. compared with the commercial dosage form, the zein-loaded emamectin benzoate nanometer slow-release agent has obviously reduced contact angle on cucumber and cabbage leaves, obviously increased retention, and can obviously improve the spreading and infiltration performance of pesticides on target crop leaves, thereby reducing drug loss and improving drug effect;
5. Compared with the commercial dosage forms, the zein-loaded emamectin benzoate nanometer slow release agent has remarkable slow release effect under the pH neutral environment, can keep slow and stable release for a long time, can perform emamectin benzoate responsive release along with the change of the environmental pH and enzyme, and is more beneficial to quickly killing lepidopteran insect larvae;
6. indoor biological activity tests and field efficacy tests show that the zein-loaded emamectin benzoate nanometer slow-release agent has obviously higher indoor toxicity effect and field efficacy on lepidopteran insects, namely asparagus caterpillar, than the commercial dosage forms, and has better insecticidal effect;
7. In terms of materials, the zein is selected as a carrier material, so that the environment is not polluted, the detention time of the medicine on the surface of the target is prolonged, and the effective utilization rate of the medicine is improved;
8. The zein-supported emamectin benzoate nanometer slow-release agent is prepared by taking zein as a carrier material and adopting a phase separation method, heating or crosslinking is not needed in the preparation process, an emulsifying agent is not needed, and only non-toxic ethanol and water are used as solvents, so that the preparation process is simple, easy to operate, good in repeatability, good in safety and small in environmental pollution.
Drawings
FIG. 1 is a schematic diagram of the digestive tract structure of lepidopteran insect larvae;
FIG. 2 shows scanning electron microscope and transmission electron microscope images of zein-loaded emamectin benzoate nano slow release formulations of the present invention;
FIG. 3 shows images of zein-loaded emamectin benzoate nano slow release formulations of the present invention after storage at different temperatures;
FIG. 4 shows the mean particle size and dispersion index (PDI) of zein-loaded emamectin benzoate nano slow release formulations of the present invention stored at different temperatures as a function of time;
FIG. 5 shows a standard curve of emamectin benzoate in 75% methanol;
FIG. 6 shows the variation of the photodegradation rate over time of emamectin benzoate (EMB) and Zein-loaded emamectin benzoate nano-release formulation (Zein-EMB-NC) of the present invention under UV irradiation;
FIG. 7 shows contact angle pictures of deionized water, commercially available emamectin benzoate water dispersible granule (EMB-WDG), commercially available emamectin benzoate microemulsion (EMB-ME) and Zein-loaded emamectin benzoate nano slow release formulation (Zein-EMB-NC) of the present invention on cucumber leaves;
FIG. 8 shows contact angle pictures of deionized water, commercially available emamectin benzoate water dispersible granule (EMB-WDG), commercially available emamectin benzoate microemulsion (EMB-ME) and Zein-loaded emamectin benzoate nano slow release formulation (Zein-EMB-NC) of the present invention on cabbage leaves;
FIG. 9 shows the contact angle comparisons of deionized water, commercial emamectin benzoate water dispersible granule (EMB-WDG), commercial emamectin benzoate microemulsion (EMB-ME), and Zein-loaded emamectin benzoate nano slow release formulation (Zein-EMB-NC) of the present invention on cucumber and cabbage leaves;
FIG. 10 shows the hold-up comparisons of deionized water, commercial emamectin benzoate water dispersible granule (EMB-WDG), commercial emamectin benzoate microemulsion (EMB-ME), and Zein-loaded emamectin benzoate nano slow release formulation (Zein-EMB-NC) of the present invention on cucumber and cabbage leaves;
FIG. 11 shows cumulative release profiles of commercial emamectin benzoate microemulsions (EMB-ME) and Zein-loaded emamectin benzoate nano slow release formulations (Zein-EMB-NC) of the present invention under different environments.
Detailed Description
The invention is described below with reference to specific examples. It will be appreciated by those skilled in the art that these examples are for illustration of the invention only and are not intended to limit the scope of the invention in any way.
The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials of the medicines, the materials of the reagents, and the like used in the examples described below are commercially available products unless otherwise specified.
Instrument for measuring and controlling the intensity of light
Magnetic stirrer (MS 7-H550-Pro DLAB)
Dynamic light scattering laser particle size analyzer (ZETASIZER NANOZS, 90 Malvern)
Scanning electron microscope (JSM-6700F JEOL)
Transmission electron microscope (CM 120 Philips)
Contact angle measuring instrument (JC 2000D 2M, shanghai middle morning)
Ultraviolet-visible spectrophotometer (TU 901, shimadzu)
Xenon arc lamp illumination incubator (XT-5409-XPC 80, xutemp)
Materials and reagents
Emamectin benzoate (70% purity, hebei Weiyuan Biochemical Co., ltd.)
Zein (national medicine group chemical reagent Co., ltd.)
Alkaline protease (Beijing Soy Bao technology Co., ltd.)
Dialysis bag (molecular weight cut-off 5000Da Beijing Tianan combined science and technology Co., ltd.)
Example 1 preparation of zein-loaded emamectin benzoate nanometer sustained release agent
50Mg of emamectin benzoate and 100mg of zein are dissolved in 20ml of ethanol water solution with the volume fraction of 70 percent, vortex oscillation is carried out for 2-4min at the speed of 300r/min, and yellow clear and transparent organic solution is obtained, wherein the concentration of the emamectin benzoate in the organic solution is 2.5g/L, and the concentration of the zein is 5g/L.
Slowly injecting 1ml of the obtained organic solution into 20ml of 37 ℃ water by using a pipetting gun, placing the solution on a magnetic stirrer to keep constant temperature, stirring for 1h at 300r/min, and volatilizing the organic solvent to obtain the zein-loaded emamectin benzoate nanometer slow-release agent.
Example 2 preparation of zein-loaded emamectin benzoate nanometer sustained release agent
100Mg of emamectin benzoate and 200mg of zein are dissolved in 20ml of ethanol water solution with the volume fraction of 70 percent, vortex oscillation is carried out for 2-4min at the speed of 300r/min, and yellow clear and transparent organic solution is obtained, wherein the concentration of the emamectin benzoate in the organic solution is 5g/L, and the concentration of the zein is 10g/L.
Slowly injecting 1ml of the obtained organic solution into 20ml of 37 ℃ water by using a pipetting gun, placing the solution on a magnetic stirrer to keep constant temperature, stirring for 1h at 300r/min, and volatilizing the organic solvent to obtain the zein-loaded emamectin benzoate nanometer slow-release agent.
Example 3 preparation of zein-loaded emamectin benzoate nanometer sustained release agent
200Mg of emamectin benzoate and 300mg of zein are dissolved in 20ml of ethanol water solution with the volume fraction of 70 percent, vortex oscillation is carried out for 2-4min at the speed of 300r/min, and yellow clear and transparent organic solution is obtained, wherein the concentration of the emamectin benzoate in the organic solution is 10g/L, and the concentration of the zein is 15g/L.
Slowly injecting 1ml of the obtained organic solution into 20ml of 37 ℃ water by using a pipetting gun, placing the solution on a magnetic stirrer to keep constant temperature, stirring for 1h at 300r/min, and volatilizing the organic solvent to obtain the zein-loaded emamectin benzoate nanometer slow-release agent.
Example 4 preparation of zein-loaded emamectin benzoate nanometer sustained release agent
50Mg of emamectin benzoate and 200mg of zein are dissolved in 20ml of ethanol water solution with the volume fraction of 70 percent, vortex oscillation is carried out for 2-4min at the speed of 300r/min, and yellow clear and transparent organic solution is obtained, wherein the concentration of the emamectin benzoate in the organic solution is 2.5g/L, and the concentration of the zein is 10g/L.
Slowly injecting 1ml of the obtained organic solution into 20ml of 37 ℃ water by using a pipetting gun, placing the solution on a magnetic stirrer to keep constant temperature, stirring for 2 hours at 300r/min, and volatilizing the organic solvent to obtain the zein-loaded emamectin benzoate nanometer slow-release agent.
Example 5 preparation of zein-loaded emamectin benzoate nanometer sustained release agent
100Mg of emamectin benzoate and 300mg of zein are dissolved in 20ml of ethanol water solution with the volume fraction of 70 percent, vortex oscillation is carried out for 2-4min at the speed of 300r/min, and yellow clear and transparent organic solution is obtained, wherein the concentration of the emamectin benzoate in the organic solution is 5g/L, and the concentration of the zein is 15g/L.
Slowly injecting 1ml of the obtained organic solution into 20ml of 37 ℃ water by using a pipetting gun, placing the solution on a magnetic stirrer to keep constant temperature, stirring for 2 hours at 300r/min, and volatilizing the organic solvent to obtain the zein-loaded emamectin benzoate nanometer slow-release agent.
Example 6 preparation of zein-Supported emamectin benzoate nanometer sustained release agent
200Mg of emamectin benzoate and 100mg of zein are dissolved in 20ml of ethanol water solution with the volume fraction of 70 percent, vortex oscillation is carried out for 2-4min at the speed of 300r/min, and yellow clear and transparent organic solution is obtained, wherein the concentration of the emamectin benzoate in the organic solution is 10g/L, and the concentration of the zein is 5g/L.
Slowly injecting 1ml of the obtained organic solution into 20ml of 37 ℃ water by using a pipetting gun, placing the solution on a magnetic stirrer to keep constant temperature, stirring for 2 hours at 300r/min, and volatilizing the organic solvent to obtain the zein-loaded emamectin benzoate nanometer slow-release agent.
Example 7 preparation of zein-loaded emamectin benzoate nanometer sustained release agent
50Mg of emamectin benzoate and 300mg of zein are dissolved in 20ml of ethanol water solution with the volume fraction of 70 percent, vortex oscillation is carried out for 2-4min at the speed of 300r/min, and yellow clear and transparent organic solution is obtained, wherein the concentration of the emamectin benzoate in the organic solution is 2.5g/L, and the concentration of the zein is 15g/L.
Slowly injecting 1ml of the obtained organic solution into 20ml of 37 ℃ water by using a pipetting gun, placing the solution on a magnetic stirrer to keep constant temperature, stirring for 3 hours at 300r/min, and volatilizing the organic solvent to obtain the zein-loaded emamectin benzoate nanometer slow-release agent.
Example 8 preparation of zein-Supported emamectin benzoate nanometer sustained release agent
100Mg of emamectin benzoate and 100mg of zein are dissolved in 20ml of ethanol water solution with the volume fraction of 70 percent, vortex oscillation is carried out for 2-4min at the speed of 300r/min, and yellow clear and transparent organic solution is obtained, wherein the concentration of the emamectin benzoate in the organic solution is 5g/L, and the concentration of the zein is 5g/L.
Slowly injecting 1ml of the obtained organic solution into 20ml of 37 ℃ water by using a pipetting gun, placing the solution on a magnetic stirrer to keep constant temperature, stirring for 3 hours at 300r/min, and volatilizing the organic solvent to obtain the zein-loaded emamectin benzoate nanometer slow-release agent.
Example 9 preparation of zein-loaded emamectin benzoate nanometer sustained release agent
200Mg of emamectin benzoate and 200mg of zein are dissolved in 20ml of ethanol water solution with the volume fraction of 70 percent, vortex oscillation is carried out for 2-4min at the speed of 300r/min, and yellow clear and transparent organic solution is obtained, wherein the concentration of the emamectin benzoate in the organic solution is 10g/L, and the concentration of the zein is 10g/L.
Slowly injecting 1ml of the obtained organic solution into 20ml of 37 ℃ water by using a pipetting gun, placing the solution on a magnetic stirrer to keep constant temperature, stirring for 3 hours at 300r/min, and volatilizing the organic solvent to obtain the zein-loaded emamectin benzoate nanometer slow-release agent.
Test 1 comparison of the Performance of zein-loaded Emamectin salt nanometer sustained release agent and sustained release agent prepared by zein-loaded other pesticide active ingredients
According to the invention, 2 nonpolar pesticides (abamectin and chlorantraniliprole) and 1 polar pesticide (dinotefuran) are selected as pesticide active ingredients, and a zein-loaded abamectin sustained release agent, a zein-loaded chlorantraniliprole sustained release agent and a zein-loaded dinotefuran sustained release agent are prepared respectively by adopting the same method as that of the example 9.
The prepared zein-loaded avermectin sustained release agent, zein-loaded chlorantraniliprole sustained release agent and zein-loaded dinotefuran sustained release agent prepared in example 9 are diluted to 0.05% (w/w) respectively by deionized water, then the zein-loaded emamectin benzoate sustained release agent is uniformly dispersed by ultrasonic oscillation for 5-10s, 2.5mL of sample solution is added into a cuvette, the cuvette is placed into a dynamic light scattering laser particle sizer, and the average hydration particle size and polydispersity index (Polydispersity index, PDI) of the four zein sustained release agents are measured at 25 ℃. The measurements were repeated 3 times for each sample and averaged. The average hydrated particle size, PDI and sample state of zein sustained release formulations prepared with different pesticide active ingredients were compared.
TABLE 1 average hydrated particle size, PDI and sample State of zein sustained release formulations prepared with different pesticide active ingredients
Table 1 shows the average hydrated particle size, PDI and sample status of zein sustained release formulations prepared with different pesticide active ingredients. From Table 1, it can be seen that the particle size of zein sustained release agent prepared from avermectin and dinotefuran is obviously larger, both exceeds 1 μm, PDI is more than 0.3, and suspended substances or bottom precipitation phenomenon is shown in the drug solution; the particle size of the zein sustained release agent prepared by chlorantraniliprole is relatively small, but PDI is more than 0.3, and a small amount of yellow precipitate exists at the bottom of the drug solution; the particle size of the zein sustained release agent prepared by emamectin benzoate is minimum, PDI is less than 0.3, the particle size distribution is uniform, and the solution is in a stable and transparent state. Therefore, the particle size, PDI and sample state of the zein sustained release agent prepared by adopting emamectin benzoate are obviously superior to those of zein sustained release agents prepared by adopting other pesticide active ingredients.
Test 2 test of drug-loading rate, encapsulation efficiency, particle size and PDI of zein-loaded emamectin benzoate nanometer sustained release agent
The drug-loading rate and the encapsulation efficiency of the zein-loaded emamectin benzoate nanometer slow-release agent prepared in the embodiments 1-9 are measured by adopting a high-speed centrifugal filtration method, and the specific method is as follows: accurately absorbing 1mL of zein loaded emamectin benzoate nanometer slow release agent, adding into 3mL of methanol solution, performing ultrasonic demulsification, filtering with a 0.22 mu m filter membrane, measuring absorbance value at 245nm by an ultraviolet-visible spectrophotometer, and calculating emamectin benzoate concentration by using a emamectin benzoate standard curve to obtain total medicine amount C 0; and (3) adding 1mL of zein loaded emamectin benzoate nanometer slow release agent into a 1.5mL centrifuge tube, centrifuging at 15000rpm for 10min, filtering the supernatant with a 0.22 mu m filter membrane, adding three times of methanol solution, measuring absorbance value at 245nm by an ultraviolet-visible spectrophotometer, and calculating emamectin benzoate concentration by using a emamectin benzoate standard curve to obtain free medicine content C 1. Drug loading (Drug Loading Content, DLC) and encapsulation efficiency (ENTRAPMENT EFFICIENCY, EE) were calculated from the following formulas, respectively:
In the above formula, M EMB and M zein represent the amounts of emamectin benzoate and zein, respectively.
The encapsulation efficiency and the drug-loading rate of the zein-loaded emamectin benzoate nanometer slow-release agent prepared in examples 1-9 are calculated according to the formula, and the test results are shown in table 2.
In addition, the particle size and PDI of the zein-loaded emamectin benzoate nanometer slow release agent prepared in examples 1-9 were obtained by testing through a dynamic light scattering laser particle sizer by the same method as in test 1, and the test results are shown in Table 2.
Table 2 particle size, PDI, encapsulation efficiency and drug loading capacity of zein-loaded emamectin benzoate nano slow-release formulations prepared in examples 1-9
From Table 2, the particle size of the zein-loaded emamectin benzoate nanometer slow release agent prepared in the embodiments 1-9 is 141.7-189.0nm, the PDI is 0.096-0.219, the encapsulation efficiency is 33.3-50.8%, and the drug loading rate is 7.0-21.3%. The particle size, PDI, encapsulation efficiency and drug loading are comprehensively considered, the particle size and PDI of the zein loaded emamectin benzoate nano slow-release agent prepared in the embodiment 8 are smaller, the encapsulation efficiency and drug loading are larger, and the performances of all aspects are better. Therefore, the method and the process parameters of the preferred embodiment 8 of the invention prepare the zein-loaded emamectin benzoate nanometer slow release agent.
Test 3 Structure and morphology characterization of zein-loaded emamectin benzoate nanometer sustained release agent
The apparent morphology of the zein-loaded emamectin benzoate nanometer slow-release agent prepared in the example 8 is observed by a scanning electron microscope and a transmission electron microscope respectively. Fig. 2 shows a scanning electron microscope and a transmission electron microscope image of the zein-loaded emamectin benzoate nano slow-release agent according to the invention, wherein fig. 2a and fig. 2b are scanning electron microscope images at 10000 times magnification, and fig. 2c and fig. 2d are transmission electron microscope images at 3000 times magnification and 6000 times magnification, respectively. As shown in figure 2, the zein-loaded emamectin benzoate nanometer slow-release agent is spherical with smooth surface and regular morphology, has smaller particle size (similar to the particle size obtained by adopting a dynamic light scattering laser particle sizer in test 2), and has better particle dispersibility.
Test of storage stability test of 4-zein-loaded emamectin benzoate nanometer sustained release agent
The storage stability test of the zein-loaded emamectin benzoate nano slow-release agent prepared in example 8 was carried out according to the International Commission on pesticide analysis (CIPAC MT 46) and the national Standard for pesticide stability determination (GB/T19136-2003), based on the pesticide Low temperature stability determination method (GB/T19137-2003), the pesticide ambient temperature storage stability test Notification (NY/T1427-2016) and the pesticide thermal storage stability determination method (GB/T19136-2003), and the specific methods are as follows: accurately absorbing 5mL zein loaded emamectin benzoate nanometer slow release agent, subpackaging into a brown reagent bottle with the volume of 20mL, sealing, placing a part of the mixture in a refrigerator with the temperature of 0+/-2 ℃ for low-temperature stability test, storing for 7 days, taking out samples after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days and 7 days respectively, observing the appearance of the samples, and measuring the particle size and PDI of the samples by adopting a dynamic light scattering laser particle sizer; and the other part is respectively placed in a room temperature dryer with the temperature of (25+/-2) ℃ and an incubator with the temperature of (54+/-2) ℃ for stability test, and is stored for 14 days, and samples are taken out after 2 days, 4 days, 6 days, 8 days, 10 days, 12 days and 14 days respectively, the appearance of the samples is observed, and the particle size and the PDI of the samples are measured by adopting a dynamic light scattering laser particle sizer.
Figure 3 shows images of zein-loaded emamectin benzoate nano slow release formulations of the present invention after storage at different temperatures. As shown in figure 3, the zein-loaded emamectin benzoate nanometer slow release agent has no obvious change in appearance after being stored at 0 ℃ for 7 days and at 25 ℃ and 54 ℃ for 14 days, keeps a clear and transparent light blue solution state, and has better storage stability.
Fig. 4 shows the mean particle size and dispersion index (PDI) of zein-loaded emamectin benzoate nano slow release formulations of the present invention stored at different temperatures as a function of time (where the bar graph represents the change in particle size and the curve represents the change in PDI). As can be seen from FIG. 4, the zein-loaded emamectin benzoate nano slow-release agent disclosed by the invention has no obvious change in average particle size and PDI after being stored at 0 ℃ for 7 days and at 25 ℃ and 54 ℃ for 14 days, and shows good physicochemical stability. Therefore, the zein-loaded emamectin benzoate nano slow-release agent can be stored at different temperatures, can keep stable and uniform dispersion, has no obvious change of particle size, has good storage stability, and is beneficial to improving the utilization rate of pesticides.
Test 5 photo stability test of zein-loaded emamectin benzoate nanometer sustained release agent
The experiment uses emamectin benzoate as a reference, adopts a xenon arc lamp illumination incubator to simulate a natural light environment in the field, and measures the photodegradation rate of the zein-loaded emamectin benzoate nano slow-release agent prepared in the example 8 by an ultraviolet-visible spectrophotometer, wherein the specific experimental method is as follows: accurately weighing 20mg of emamectin benzoate crude drug and 4mL of zein-loaded emamectin benzoate nanometer slow release agent, respectively placing into a transparent plastic culture dish, adding 4mL of deionized water into the emamectin benzoate crude drug to uniformly disperse, and setting 6 repetitions for each sample. Then, the petri dish was placed in a xenon arc lamp illumination incubator, and an ultraviolet lamp (emission wavelength: 365 nm) was set to continuously irradiate at 500W and a temperature of 25 degrees for 72 hours, and samples were taken at 12 hours, 24 hours, 36 hours, 48 hours, 60 hours and 72 hours, respectively. Adding 75% methanol solution into the taken emamectin benzoate raw medicine sample, transferring to a 50mL volumetric flask for constant volume, measuring absorbance value at 245nm by using an ultraviolet-visible spectrophotometer, and calculating the concentration of the emamectin benzoate according to an emamectin benzoate standard curve (shown in figure 5), thereby obtaining the photodegradation rate of the emamectin benzoate raw medicine. In addition, 75% methanol solution is added into the taken zein-loaded emamectin benzoate nanometer slow-release agent sample, then the zein-loaded emamectin benzoate nanometer slow-release agent sample is transferred into a 50mL volumetric flask for constant volume, the emamectin benzoate content of the zein-loaded emamectin benzoate nanometer slow-release agent sample is measured by adopting a high-speed centrifugal filtration method, and the emamectin benzoate concentration is calculated according to a emamectin benzoate standard curve (shown in figure 5), so that the photodegradation rate of the zein-loaded emamectin benzoate nanometer slow-release agent is obtained.
FIG. 6 shows the variation of the photodegradation rate over time of emamectin benzoate (EMB) and Zein-loaded emamectin benzoate nano slow release formulation (Zein-EMB-NC) of the present invention under UV irradiation. As shown in FIG. 6, the emamectin benzoate technical is particularly sensitive to illumination, and the degradation rate reaches 42.9% when the illumination is carried out for 24 hours, but only 19.2% of emamectin benzoate in the zein-loaded emamectin benzoate nanometer slow release agent is degraded at the moment; the degradation rate of the emamectin benzoate original drug reaches 52.4% when the light is irradiated for 36 hours, and the degradation rate of the zein-loaded emamectin benzoate nanometer slow release agent is only 23.6%; the degradation rate of the emamectin benzoate raw medicine is up to 72.4% at 72h, and the degradation rate of the zein-loaded emamectin benzoate nano slow-release agent is only 26.8%, which is 2.7 times of that of the zein-loaded emamectin benzoate nano slow-release agent. Therefore, the zein loaded emamectin benzoate nanometer slow-release agent can effectively reduce the photodegradation rate of the pesticide active ingredient emamectin benzoate, obviously improve the photostability of the emamectin benzoate under the field illumination condition, greatly reduce the decomposition loss of the emamectin benzoate, the zein carrier is used as a shell layer to protect the emamectin benzoate from being contacted with external environment ultraviolet rays, and the zein has good ultraviolet light resisting effect, improves the light stability and effectively prolongs the drug effect time.
Blade contact angle measurement for testing 6 zein loaded emamectin benzoate nanometer slow release agent
The contact angle of the zein-loaded emamectin benzoate nanometer slow release agent, the commercially available emamectin benzoate water dispersible granule and the commercially available emamectin benzoate microemulsion prepared in the example 8 on the hydrophilic leaf (cucumber) and the hydrophobic leaf (cabbage) is measured by a contact angle measuring instrument. The specific experimental steps are as follows: the zein loaded emamectin benzoate nanometer slow release agent, the commercial emamectin benzoate water dispersible granule and the commercial emamectin benzoate microemulsion are respectively diluted to 0.05% (w/w) by deionized water, and are uniformly vibrated by ultrasound for standby. Healthy cucumber leaves and purchased fresh cabbage leaves grown in a climatic incubator (430D) were then collected, gently rinsed with deionized water to remove surface impurities, and after natural air-drying, the elongated portions between the leaf veins of the leaves were cut as the subject, and fixed flat with double sided tape on a clean glass slide (note that the whole process did not destroy the structural morphology of the leaves). Finally, placing the glass slide on the objective table, adjusting the heights of the injection needle head and the objective table and the focal length of the lens to enable the side surface of the blade and the needle head to be clearly displayed in the visual field of the screen, setting the sample injection amount to be 5 mu L, immediately capturing an image when the blade and the liquid drop are stably contacted, analyzing the contact angle of the liquid medicine and the blade by using a five-point fitting method, and measuring for multiple times to obtain an average value. The experiment uses clear water as a control.
Fig. 7 and 8 show contact angle pictures of deionized water, commercially available emamectin benzoate water dispersible granule (EMB-WDG), commercially available emamectin benzoate microemulsion (EMB-ME), and Zein-loaded emamectin benzoate nano slow release formulation (Zein-EMB-NC) of the present invention on cucumber and cabbage leaves, respectively. Fig. 9 shows the contact angle comparisons of deionized water, commercial emamectin benzoate water dispersible granule (EMB-WDG), commercial emamectin benzoate microemulsion (EMB-ME), and Zein-loaded emamectin benzoate nano slow release formulation (Zein-EMB-NC) of the present invention on cucumber and cabbage leaves. As shown in fig. 7-9, the contact angles of deionized water, commercial emamectin benzoate water dispersible granule (EMB-WDG), commercial emamectin benzoate microemulsion (EMB-ME) and Zein-EMB-NC of the invention on cucumber leaves are respectively 95.2 °, 78.9 °, 68.9 ° and 55.6 °, and the contact angles of the Zein-EMB-NC on cabbage leaves are respectively 104.6 °, 80.1 °, 65.9 ° and 63.5 °, respectively, the contact angles of the Zein-loading emamectin benzoate nano slow release preparation of the invention on cucumber and cabbage leaves are obviously smaller than those of the commercial emamectin benzoate water dispersible granule and the commercial emamectin benzoate microemulsion, and the difference is obvious (as shown in fig. 9, wherein ". X" indicates that when P < 0.01), the difference is extremely obvious, the smaller the contact angle of the leaf surface is, the Zein-loading emamectin benzoate nano slow release preparation of the invention indicates that the liquid medicine is easy to spread and infiltrate on the leaf surface, so that the spreading and the pesticide effect of the pesticide on the leaf surface of the crop can be obviously reduced, and the drug effect of the target drug is obviously improved.
Test 7 leaf retention measurement of zein-loaded emamectin benzoate nanometer slow-release agent
The retention of the zein-loaded emamectin benzoate nanometer slow-release agent, the commercially available emamectin benzoate water dispersible granule and the commercially available emamectin benzoate microemulsion prepared in the example 8 on hydrophilic leaves (cucumbers) and hydrophobic leaves (cabbages) is measured in the experiment. The specific experimental steps are as follows: the zein loaded emamectin benzoate nanometer slow release agent, the commercial emamectin benzoate water dispersible granule and the commercial emamectin benzoate microemulsion are respectively diluted to 0.05% (w/w) by deionized water, and are uniformly vibrated by ultrasound for standby. Then, collecting healthy cucumber leaves and purchased fresh cabbage leaves grown in an artificial climate incubator (430D), lightly flushing the fresh cabbage leaves with deionized water to remove surface impurities, preparing round regular leaves (avoiding main veins as much as possible) by a puncher with the diameter of 1cm after naturally airing, and measuring to obtain leaf area S (cm 2) and initial mass M 0; soaking the leaf in the liquid medicine for 15s, vertically taking out, suspending for 30s until no liquid drops flow down, and weighing the mass M 1; finally, the difference in mass of the blade before and after the treatment was calculated, and the blade retention R m was calculated according to the following formula, and each sample was measured 3 times to average. The experiment uses clear water as a control.
FIG. 10 shows the retention comparisons of deionized water, commercial emamectin benzoate water dispersible granule (EMB-WDG), commercial emamectin benzoate microemulsion (EMB-ME), and Zein-loaded emamectin benzoate nano slow release formulation (Zein-EMB-NC) of the present invention on cucumber and cabbage leaves. As seen in FIG. 10, the retention amounts of clear water on the cucumber leaf and cabbage leaf were 23.35mg/cm 2 and 14.46mg/cm 2, respectively, indicating that the cucumber leaf has better hydrophilic properties than the cabbage leaf. The retention amounts of Zein-loaded emamectin benzoate nanometer slow release agent (Zein-EMB-NC), commercial emamectin benzoate microemulsion (EMB-ME) and commercial emamectin benzoate water dispersible granule (EMB-WDG) on cucumber leaves are 46.80mg/cm 2、38.86mg/cm2、35.95mg/cm2 and 21.95mg/cm 2、18.79mg/cm2、18.05mg/cm2 respectively; therefore, the retention of the zein-loaded emamectin benzoate nano slow-release agent on cucumber and cabbage leaves is higher than that of the commercial emamectin benzoate microemulsion and the commercial emamectin benzoate water dispersible granule, the difference is obvious (as shown in figure 10, wherein the difference is very obvious when P is less than 0.01, and the difference is obvious when P is less than 0.05), so that the zein-loaded emamectin benzoate nano slow-release agent has good wettability, thereby being beneficial to reducing the loss of medicines and improving the efficacy. Test of sustained release performance of 8 zein-loaded emamectin benzoate nanometer sustained release agent
The in-vitro release behavior of the zein-loaded emamectin benzoate nanometer slow-release agent prepared in the example 8 is measured by a dialysis bag method, and the response release capacity of the zein-loaded emamectin benzoate nanometer slow-release agent to pH and enzyme is tested by simulating the environment in the alkaline intestinal tract of lepidopteran insects. First, 4 groups of samples were prepared: ① Cutting a dialysis bag for a certain length, flushing with deionized water, accurately weighing commercially available emamectin benzoate microemulsion (EMB-ME), placing into the flushed dialysis bag, and adding 10mL of deionized water to uniformly disperse (the sample mark is EMB-ME (pH=7)); ② Intercepting a dialysis bag for a certain length, flushing the dialysis bag with deionized water, accurately weighing 10mL of Zein-loaded emamectin benzoate nanometer slow release agent, and placing the slow release agent in the flushed dialysis bag (the sample is marked as Zein-EMB-NC (pH=7)); ③ Intercepting a dialysis bag for a certain length, flushing the dialysis bag with deionized water, accurately weighing 10mL of Zein-loaded emamectin benzoate nanometer slow release agent, placing the slow release agent into the flushed dialysis bag, and adding a proper amount of NaOH to adjust the pH to 9 (a sample is marked as Zein-EMB-NC (pH=9)); ④ Cutting a dialysis bag for a certain length, flushing with deionized water, accurately weighing 10mL of Zein-loaded emamectin benzoate nanometer slow release agent, placing into the flushed dialysis bag, adding a proper amount of NaOH to adjust the pH to 9, and then adding alkaline protease (the sample is marked as Zein-EMB-NC (pH=9+enzyme)). Then, both ends of the 4 groups of samples were sealed with a clip and placed in 150mL brown reagent bottles, and 40mL of 20% aqueous methanol solution was supplemented to 50mL, so that the dialysis bag was immersed therein. Finally, the bottle mouth is sealed and transferred to a constant temperature shaking table, shaking dialysis is carried out at 25 ℃ and 300r/min, 3mL of dialysate is taken from the bottle at regular time for testing, and the same volume of fresh slow-release medium (20% methanol water solution) is immediately replenished, so that the total volume is always 50mL.
And measuring the absorbance value of the obtained dialysate by using an ultraviolet-visible spectrophotometer at 245nm, calculating the accumulated release amount of the emamectin benzoate in the slow-release solution at different times according to an emamectin benzoate standard curve, and drawing an accumulated release curve.
FIG. 11 shows cumulative release profiles of commercial emamectin benzoate microemulsions (EMB-ME) and Zein-loaded emamectin benzoate nano slow release formulations (Zein-EMB-NC) of the present invention under different environments. As shown in fig. 11, the cumulative release amount of the zein-loaded emamectin benzoate nano slow release agent is only 37.7% in the 250h environment with neutral pH, and the slow and stable release can be maintained for a long time; whereas the accumulated release amount of the commercial emamectin benzoate microemulsion (EMB-ME) under the pH neutral environment for 100 hours is 74 percent, the accumulated release amount under 250 hours is 94.5 percent, compared with commercial emamectin benzoate microemulsion, the zein-loaded emamectin benzoate nanometer slow release agent has remarkable slow release effect under the neutral pH environment. Compared with a pH neutral environment, the zein-loaded emamectin benzoate nanometer slow release agent has the advantages that the emamectin benzoate release rate in a pH alkaline environment (simulating lepidopteran insect alkaline intestinal environment (pH=9)) is increased to a certain extent, the accumulated release rate reaches 51.7% in 250 hours, after alkaline protease is further added in the alkaline environment, the emamectin benzoate release rate is obviously increased, the emamectin benzoate accumulated release rate reaches 50% in 50 hours, the emamectin benzoate accumulated release rate reaches 87% in 100 hours, and the emamectin benzoate accumulated release rate reaches 93.7% in 250 hours, and the zein-loaded emamectin benzoate nanometer slow release agent has obvious enzyme response release characteristics.
In conclusion, compared with the commercial emamectin benzoate microemulsion, the zein-loaded emamectin benzoate nanometer slow release agent has remarkable slow release effect under the neutral pH environment, can keep slow and stable release for a long time, has the emamectin benzoate release rate higher than that in the neutral environment in the alkaline environment with pH of 9, still presents stable and slow release, has remarkably increased emamectin benzoate release rate after alkaline protease is added, and has good enzyme stimulation response performance. Therefore, after the zein-loaded emamectin benzoate nanometer slow-release agent enters the body of the pests, the release of the active ingredients of the emamectin benzoate can be accelerated under the dual actions of alkaline environment and alkaline protease, and the pests can be killed quickly.
Indoor bioactivity assay for testing 9 zein-loaded emamectin benzoate nanometer slow release agent
Pesticide according to the "pesticide indoor bioassay test guidelines" section 9: spray method (NY/T1154.9-2008), the indoor biological activities of Zein-loaded emamectin benzoate nanometer slow release agent (Zein-EMB-NC), three commercial control formulations (commercial emamectin benzoate water dispersible granule (EMB-WDG), commercial emamectin benzoate microemulsion (EMB-ME) and commercial emamectin benzoate water soluble granule (EMB-SG)) prepared in the embodiment 8 of the invention are measured by adopting a spray method. The specific method comprises the following steps: the zein-loaded emamectin benzoate nanometer slow release agent and three commercial control dosage forms are respectively prepared into 6 concentration gradients by purified water in equal proportion, the concentration of the agents is respectively set to 10, 5, 2.5, 1.25, 0.625 and 0.3125mg/L, and corresponding treatments without the agents are set as blank controls; cutting artificial feed into slices, placing the slices in a plastic culture dish with the diameter of 6cm, lightly picking 3-year-old larvae of asparagus caterpillar on the artificial feed by using a writing brush, placing 15 heads of the larvae on each dish, then placing under a Bode spray tower for accurate spraying, spraying the liquid medicine amount of 2mL each time, spraying the liquid medicine under 68.9kPa, settling for 30s after each spraying, and then placing in a constant-temperature incubator with the temperature of 25 ℃ for feeding, wherein each treatment is repeated for 3 times.
After 48h and 72h of application, the death condition of the asparagus caterpillar is checked under an anatomical microscope, the physical active person is judged to be living insects, the body of the asparagus caterpillar is touched by a writing brush when the body of the asparagus caterpillar is difficult to distinguish, no responders are judged to be dead, and the death number is counted. Finally, a virulence regression equation, a semi-lethal concentration (LC 50) and a corresponding 95% confidence interval are calculated, and then a variance analysis and regression analysis are performed.
TABLE 3 virulence regression equation, semi-lethal concentration (LC 50) and corresponding 95% confidence interval for 48h after Zein-loaded emamectin benzoate nano slow release formulation (Zein-EMB-NC) and commercial control formulation (EMB-WDG, EMB-ME, EMB-SG) of the present invention are administered
TABLE 4 virulence regression equation, semi-lethal concentration (LC 50) and corresponding 95% confidence interval for Zein-loaded emamectin benzoate nano slow release formulation (Zein-EMB-NC) and commercial control formulation (EMB-WDG, EMB-ME, EMB-SG) of the present invention 72h post-administration
Tables 3 and 4 show the toxicity regression equations, semi-lethal concentration (LC 50) and corresponding 95% confidence intervals of zein-loaded emamectin benzoate nano slow release formulations and commercial control formulations of the present invention at 48h, 72h post-administration, respectively. As can be seen from tables 3 and 4, the LC50 of the three commercial formulations EMB-WDG, EMB-ME, EMB-SG were not significantly different at 48h post-administration, 4.72 μg/mL, 4.62 μg/mL, 4.86 μg/mL, respectively; the LC50 of the Zein-loaded emamectin benzoate nanometer slow release agent (Zein-EMB-NC) is 3.43 mug/mL, which is obviously smaller than the LC50 of other three commercial dosage forms, and the sensitivity is strongest. The LC50 of the three commercial formulations EMB-WDG, EMB-ME and EMB-SG were not greatly different at 72h after administration, and were 4.50. Mu.g/mL, 4.55. Mu.g/mL and 4.28. Mu.g/mL, respectively; the LC50 of the Zein-loaded emamectin benzoate nanometer slow release agent (Zein-EMB-NC) is 3.11 mug/mL, which is obviously smaller than the LC50 of other three commercial formulations, has the strongest sensibility and obviously better toxicity effect on asparagus caterpillar than the emamectin benzoate commercial formulations.
In conclusion, compared with the commercial dosage form of the emamectin benzoate, the semi-lethal concentration (LC 50) of the zein-loaded emamectin benzoate nano slow-release agent is obviously reduced, and the toxicity effect on lepidoptera insects, namely asparagus caterpillar is obviously improved. The zein-loaded emamectin benzoate nanometer slow-release agent disclosed by the invention exists in a form of nanometer microcapsule, and the active ingredient of the original drug emamectin benzoate is not completely encapsulated, so that the active ingredient which is not encapsulated after being sprayed rapidly plays a role in killing insects, and the zein-loaded emamectin benzoate nanometer slow-release agent disclosed by the invention has quick-acting property; the encapsulated emamectin benzoate active ingredient is released after entering the alkaline intestinal tract of the insect due to the decomposition of the zein shell, so that the zein-loaded emamectin benzoate nano slow-release agent has good persistence.
Field efficacy test for testing 10 zein loaded emamectin benzoate nanometer slow release agent
In order to test the field application effect of the zein-loaded emamectin benzoate nanometer slow-release agent on asparagus caterpillar, the inventor performs a field efficacy test in peanut fields in Hebei province in 2020, wherein the test crops are peanuts, and the test is provided with 6 agent treatments: a: the zein loaded emamectin benzoate nanometer slow release agent prepared in the embodiment 8 has the concentration of 0.05 percent, and the application rate (the effective component amount) is 4.5g/hm 2; b: the zein loaded emamectin benzoate nanometer slow release agent prepared in the embodiment 8 has the concentration of 0.05 percent, and the application rate (the effective component amount) is 5.5g/hm 2; c: the zein loaded emamectin benzoate nanometer slow release agent prepared in the embodiment 8 has the concentration of 0.05 percent, and the application rate (the effective component amount) is 7.5g/hm 2; d: a dilution of commercial emamectin benzoate microemulsion at a concentration of 5.7% and a dosage (active ingredient amount) of 7.5g/hm 2; e: a dilution of commercial emamectin benzoate soluble powder at a concentration of 5.7%, a dosage (active ingredient amount) of 7.5g/hm 2; CK: blank control clear water. Each treatment is repeated for 3 times, 18 cells are arranged at random, the area of each cell is 20m 2, fertilizer and water management is consistent, and water leaf surface spraying is carried out; the test field is loam, the soil is fertile, and the irrigation is convenient. The treatment conditions of each agent are shown in table 5.
Drug effect investigation: the number of insect population was investigated 4 times before the first spraying, followed by 7, 14 and 21 days after the application, respectively. The number of the peanuts with 10 clusters is about 50 per 5 points of each community marking survey, the naturally occurring insects in the field are insufficient, the insects are complemented by the raised larvae, and the larvae are 2-3 years old.
TABLE 5 dosing rate of each agent
The efficacy was calculated according to the following formula:
the rate of reduction of insect population (%) = (pre-treatment zone number of drug-post-treatment zone number of residual insects)/pre-treatment zone number of drug-100
Correction control effect (%) = (treatment area rate of reduction of insect population-reduction of white area)/(100-reduction of insect population-reduction of white area) ×100
TABLE 6 control efficacy of each agent field test
Note that: "aA" in the table indicates that the difference in control was significant at significant levels of 5% and 1%.
The field test control of each formulation is shown in table 6. From Table 6, the zein-loaded emamectin benzoate nanometer slow release agent disclosed by the invention is sprayed in the dosage of 4.5 g/hectare, 5.5 g/hectare and 7.5 g/hectare, the prevention effect after 7 days of application reaches 92.5%, 93.9% and 97.9% respectively, and the prevention effect gradually increases with the increase of the application amount. The controlled-release agent of zein-loaded emamectin benzoate nano-emulsion, the commercial emamectin benzoate micro-emulsion and the commercial emamectin benzoate soluble powder respectively reach 97.9%, 94.8% and 94.8% in 7 days after 7.5 g/hectare application, respectively reach 98.0%, 93.9% and 93.9% in 14 days after application, respectively reach 100.0%, 84.6% and 89.7% in 21 days after application, so that the controlled-release agent of zein-loaded emamectin benzoate nano-emulsion is higher in controlled-release effect than the commercial emamectin benzoate micro-emulsion and the commercial emamectin benzoate soluble powder in 7 days, 14 days and 21 days after application under the condition of the same drug application, and the controlled-release agent has obvious difference; in addition, along with the extension of the time after the application, the prevention effect of the zein-loaded emamectin benzoate nanometer slow-release agent is better improved, and the prevention effect of different application dosage treatments has no obvious difference on the obvious level of 5% and 1%, so that the zein-loaded emamectin benzoate nanometer slow-release agent has better field insecticidal effect.
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only and are not intended to limit the scope of the present invention. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.
Claims (12)
1. The zein-loaded emamectin benzoate nanometer slow release agent is prepared from zein, emamectin benzoate, ethanol and water, and can carry out emamectin benzoate response type release along with the change of pH and/or enzyme in the environment;
The preparation method of the zein-loaded emamectin benzoate nanometer slow-release agent comprises the following steps of:
Step a: dissolving zein and emamectin benzoate in ethanol water solution, and carrying out vortex oscillation at the speed of 300r/min for 2-4min to obtain an organic solution;
Step b: c, injecting the organic solution obtained in the step a into water at 37 ℃, stirring for 3 hours at a constant temperature and 300r/min to volatilize the organic solvent, obtaining the zein-loaded emamectin benzoate nanometer slow release agent,
Wherein the volume fraction of ethanol in the ethanol aqueous solution in the step a is 70%;
the concentration of zein in the organic solution obtained in the step a is 5g/L;
the concentration of emamectin benzoate in the organic solution obtained in the step a is 5g/L;
in the organic solution obtained in the step a, the weight ratio of emamectin benzoate to zein is 1:1;
In the step b, the volume ratio of the organic solution to the water is 1:20.
2. The zein-loaded emamectin benzoate nano slow release agent according to claim 1, wherein the zein-loaded emamectin benzoate nano slow release agent has a higher emamectin benzoate release rate in a pH alkaline environment than in a pH neutral environment.
3. The zein-loaded emamectin benzoate nano slow release agent according to claim 1, wherein the zein-loaded emamectin benzoate nano slow release agent can perform emamectin benzoate response type release along with the change of alkaline protease in the environment.
4. The zein-loaded emamectin benzoate nano slow release formulation of claim 1, wherein the zein-loaded emamectin benzoate nano slow release formulation has an increased emamectin benzoate release rate in an alkaline protease environment compared to an environment in which alkaline protease is not present.
5. The zein-loaded emamectin benzoate nanometer slow release agent according to claim 1, wherein the zein-loaded emamectin benzoate nanometer slow release agent has an encapsulation rate of 33.3-50.8% and a drug loading rate of 7-21.3%.
6. The zein-loaded emamectin benzoate nano slow release agent according to claim 5, wherein the zein-loaded emamectin benzoate nano slow release agent has an encapsulation rate of 45.6% and a drug loading rate of 21.3%.
7. The zein-loaded emamectin benzoate nanometer slow release agent according to claim 1, which is characterized in that the particle size of the zein-loaded emamectin benzoate nanometer slow release agent is 100-200nm.
8. The method for preparing the zein-loaded emamectin benzoate nanometer slow release agent according to any one of claims 1 to 7, comprising the following steps:
Step a: dissolving zein and emamectin benzoate in an ethanol aqueous solution to obtain an organic solution;
Step b: c, injecting the organic solution obtained in the step a into water to volatilize the organic solvent, so as to obtain the zein-loaded emamectin benzoate nanometer slow release agent.
9. The method according to claim 8, wherein the organic solution in the step a is a yellow clear transparent organic solution.
10. The method according to claim 8, wherein in the step b, the organic solution obtained in the step a is injected into water by a pipette.
11. Use of a zein-loaded emamectin benzoate nano slow release agent according to any one of claims 1-7 as an insecticide for the control of lepidopteran insects.
12. The use according to claim 11, wherein the lepidopteran insect is asparagus caterpillar.
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