CN114467963A - Pesticide compound drug-loaded microsphere for honeysuckle and preparation method thereof - Google Patents
Pesticide compound drug-loaded microsphere for honeysuckle and preparation method thereof Download PDFInfo
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- CN114467963A CN114467963A CN202210154716.1A CN202210154716A CN114467963A CN 114467963 A CN114467963 A CN 114467963A CN 202210154716 A CN202210154716 A CN 202210154716A CN 114467963 A CN114467963 A CN 114467963A
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- caryophyllene
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- matrine
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- A01N31/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic oxygen or sulfur compounds
- A01N31/08—Oxygen or sulfur directly attached to an aromatic ring system
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- 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
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- A—HUMAN NECESSITIES
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- 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
- A01N25/12—Powders or granules
- A01N25/14—Powders or granules wettable
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- 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
- A01N27/00—Biocides, pest repellants or attractants, or plant growth regulators containing hydrocarbons
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- 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
- A01N35/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical
- A01N35/02—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical containing aliphatically bound aldehyde or keto groups, or thio analogues thereof; Derivatives thereof, e.g. acetals
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- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
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Abstract
The invention belongs to the technical field of pesticides, and discloses a compound pesticide carrying microsphere for honeysuckle and a preparation method thereof. According to the invention, the single-dose aphidicide activity is enhanced through compounding, the hollow silicon dioxide microspheres are used for loading the compound preparation to prepare the slow-release drug-carrying microspheres, the quick action, the slow release property and the lasting effect of controlling honeysuckle aphids are improved, the drug release can be matched with the aphid occurrence rule, and the effect of inhibiting the aphid population growth is achieved.
Description
The scheme is a divisional application, and the invention name of the original application is as follows: an insecticide compound drug-loaded microsphere for honeysuckle and a preparation method thereof, the application date of the original application is as follows: 2021-07-01, the application number of the original application is: 202110742337. X.
Technical Field
The invention belongs to the technical field of pesticides, and particularly relates to a pesticide compound drug-loaded microsphere for honeysuckle and a preparation method thereof.
Background
At present, honeysuckle Lonicera japonica Thunb is one of bulk drugs, and the high-quality and high-efficiency cultivation of the honeysuckle Lonicera japonica Thunb is particularly important for the development of the traditional Chinese medicine industry. The pest and disease damage phenomenon frequently occurs in the honeysuckle planting process, wherein aphids are one of the most common pests with the greatest harmfulness, the reproductive capacity is strong, the phenomenon of generation overlapping is prominent, and the characteristic of periodic parthenogenesis exists. The aphids live in the form of sucking honeysuckle juice, so that leaves and flower buds are withered and yellow and even fall off, the secreted honeydew can cause leaf sooty mold, the photosynthesis of the honeysuckle is reduced, and the yield and the quality of the honeysuckle are seriously influenced.
Honeysuckle is a common traditional Chinese medicinal material with homology of medicine and food, the demand is increased year by year, good economic benefits are achieved, and large-scale planting is carried out in many areas. In actual planting, honeysuckle diseases and insect pests frequently occur, the prevention and treatment work input is high, the effect is not ideal, and the bottleneck restricting the development of the honeysuckle industry is formed. The aphids are the most common pests on the honeysuckle and have serious harm. In the aphid control process, problems caused by large proportion and unreasonable use of chemical pesticides are increasingly prominent, such as pesticide residues, pest resistance, soil and water body pollution and the like. Compared with organic synthetic pesticides, plant-derived substances have been widely favored by researchers in recent years as resources and lead compounds for developing plant-derived pesticides due to their advantages of various modes of action, difficulty in drug resistance of pests, low toxicity to non-target organisms, and the like. But the practical application has the disadvantages of slow effect, short duration and single dosage form. How to develop a novel plant-derived pesticide with high efficiency, safety, greenness and lasting effect becomes a hotspot of current research.
The problems and defects of the prior art are as follows:
(1) in the aphid control process, the proportion of chemical pesticides is large, and due to unreasonable use, the problems of pesticide residue, pest resistance, soil and water body pollution and the like are caused; in practical application, the botanical pesticide has the disadvantages of slow effect, short duration and single dosage form.
(2) The existing plant source substances have few varieties, low yield and higher application cost; compared with chemical pesticides, the plant-derived substances have the defects of single active structure, slow response, larger influence of natural factors and the like, and have lower application rate in actual production; meanwhile, the mechanism of the plant source insecticidal substance is not clear at present, and the registered products are few in quantity and single in variety.
(3) In the prior art, the research and development results of new formulations such as screening and synergism of plant source insecticidal substances, nano sustained-release agents and the like are remarkable, such as 3.6% of nicotine and matrine microcapsule suspending agent, 3% of Microcare and the like, but the research and the report on the prevention and treatment of honeysuckle aphids are not found.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides an insecticide compound drug-loaded microsphere for honeysuckle and a preparation method thereof.
The invention is realized in such a way that the compound synergistic composition is any one of thymol, beta-caryophyllene, matrine or trans-cinnamaldehyde and azadirachtin; wherein the mass ratio of thymol to beta-caryophyllene is 0.75:1-12:1, the mass ratio of beta-caryophyllene to matrine is 1:1-16:1, and the mass ratio of trans-cinnamaldehyde to azadirachtin is 0.45:1-4.2: 1.
Further, the mass ratio of thymol, beta-caryophyllene is 3:4, 9:7, 2:1, 3:1, 9:2, 7:1, 12:1 or 2.2: 1; the mass ratio of the beta-caryophyllene and the matrine is 1:1, 12:7, 8:3, 4:1, 6:1, 28:3, 16:1 or 2.4: 1; the mass ratio of the trans-cinnamaldehyde to the azadirachtin is 9:20, 27:35, 6:5, 9:5, 27:10, 21:5 or 1.6: 1.
Further, the thymol, beta-caryophyllene and thymol beta-caryophyllene compound agent is 16% in mass percentage, the thymol is 11% in mass percentage, and the beta-caryophyllene is 5% in mass percentage; the beta-caryophyllene and matrine compound agent is 17% by mass, the beta-caryophyllene and matrine compound agent is 12% by mass and the matrine compound agent is 5% by mass; the trans-cinnamic aldehyde-azadirachtin compound is 13% by mass of the trans-cinnamic aldehyde-azadirachtin compound, the trans-cinnamic aldehyde is 8% by mass of the trans-cinnamic aldehyde-azadirachtin compound, and the azadirachtin is 5% by mass of the trans-cinnamic aldehyde-azadirachtin compound.
The invention also aims to provide the pesticide compound drug-loaded microsphere applying the compound synergistic composition, and the effective component of the pesticide compound drug-loaded microsphere is the compound synergistic composition.
The invention also aims to provide a preparation method of the pesticide compound drug-loaded microspheres, which comprises the following steps:
step one, preparing hollow silica microspheres HMSN;
step two, amino-modified hollow silica microsphere NH is carried out2-preparation of HMSN;
step three, carrying out beta-caryophyllene-matrine @ NH2Preparation of HMSN drug-loaded microspheres.
Further, in the first step, the preparation of the hollow silica microsphere HMSN comprises:
(1) weighing a certain amount of hexadecyl trimethyl ammonium bromide, dissolving the hexadecyl trimethyl ammonium bromide in an ethanol solution, magnetically stirring, adding concentrated ammonia water, fully mixing, dropwise adding tetraethyl orthosilicate, and reacting for 6 hours at room temperature;
(2) washing with deionized water and anhydrous ethanol for 3 times, centrifuging, dispersing the white precipitate into mixed solution, sealing, placing in oven for 3d at 60 deg.C, vacuum filtering, washing with deionized water for 3 times, and freeze drying;
(3) placing into a muffle furnace, calcining at 550 deg.C for 6h, removing hexadecyl trimethyl ammonium bromide, and storing at room temperature under drying condition, and recording as HMSN.
Further, in the step (1), the dosage of tetraethyl orthosilicate is 2mL, the dosage of hexadecyl trimethyl ammonium bromide is 0.15-0.3mL, and the dosage of concentrated ammonia water is 1-2 mL.
Further, in the third step, the beta-caryophyllene matrine @ NH2-preparation of HMSN drug loaded microspheres comprising:
(1) precisely weighing 100mg of beta-caryophyllene and matrine with the content of 17%, adding a proper amount of ethanol, carrying out ultrasonic treatment until the medicament is dissolved, preparing a liquid medicine with the mass concentration of 5mg/L, and heating in a water bath at 60 ℃;
(2) according to beta-caryophyllene, matrine and NH2-adding NH into HMSN with the mass ratio of 1:1-2:12Carrying out ultrasonic treatment for 30min to fully disperse HMSN, stirring for 3h in a sealed condition, continuously stirring for 1h in an open condition, and volatilizing an organic solvent;
(3) washing residual agent with hot ethanol, and freeze drying to obtain beta-caryophyllene-matrine @ NH with different proportions2-HMSN drug loaded microspheres.
Further, in the third step, the content of beta-caryophyllene matrine: NH (NH)2-HMSN mass ratio of 1: 1.
the invention also aims to provide application of the pesticide compound drug-loaded microspheres in prevention and treatment of honeysuckle aphids.
By combining all the technical schemes, the invention has the advantages and positive effects that: according to the pesticide compound drug-loaded microsphere provided by the invention, honeysuckle aphids are used as test insects, honeysuckle is used as an experimental material, plant source substances with high activity and wide effect are screened, the aphid killing activity of a single dose is enhanced through compounding, the slow-release drug-loaded microsphere is prepared, the quick acting performance and the high efficiency of the plant source liquid for controlling the aphids can be improved, the lasting period is prolonged, the harm of the aphids to the honeysuckle is reduced, a foundation is laid for green and high-efficiency control of the honeysuckle aphids, a reference way is provided for reducing the application and increasing the effect of pesticides, and the pesticide compound drug-loaded microsphere has important significance for high-quality and high-efficiency cultivation of Chinese medicinal materials.
The invention takes 17 percent beta-caryophyllene and matrine with obvious control effect as an object, uses hollow silicon dioxide microspheres to load a compound agent to prepare the slow-release drug-carrying microspheres, selects honeysuckle leaves as a target model, and researches the representation, the slow-release performance, the synergistic mechanism and the like of the drug-carrying microspheres. The results are as follows:
(1) the invention obtains the beta-caryophyllene-matrine @ NH by experiments2The optimal conditions of the HMSN drug-loaded microspheres are as follows: 0.3000g CTAB is dissolved in the mixed solution (60mL of absolute ethyl alcohol and 100mL of deionized water), the mixture is magnetically stirred, 2mL of strong ammonia water is added, 2mL of TEOS is dropwise added after the mixture is fully mixed, and the reaction is carried out for 6 hours at room temperature. And (4) reaming the washed and centrifuged white precipitate by cyclohexane, and calcining by a muffle furnace to obtain the HMSN. After amino modification, according to the proportion of beta-caryophyllene-matrine: NH (NH)2-HMSN mass ratio of 1:1 preparing medicine carrying microsphere, and the medicine carrying amount is about 36.13% under the condition. The drug-loaded microspheres have obvious slow release effect, the cumulative release rate after 288 hours is more than 80 percent, the slow release of the drug can be realized, and the drug duration is prolonged.
(2) The pharmacodynamic test shows that the beta-caryophyllene-matrine @ NH2The HMSN has good quick-acting performance and high drug effect in preventing and treating honeysuckle aphids, can continuously exert the drug effect, and can effectively inhibit the honeysuckle aphid population growth. The correction control effect of the drug-loaded microspheres after being applied for 10 days is more than 90 percent, the drug-loaded microspheres have no obvious difference compared with 10 percent imidacloprid missible oil, the correction control effect can reach 96 percent after being applied for 15 days under 500 times of liquid, and the correction control effect is obviously higher than that of beta-caryophyllene-matrine and 10 percent imidacloprid missible oil. According to the slow release characteristics of the drug-loaded microspheres, the drug-loaded microspheres are recommended to be used when the population base numbers of honeysuckle aphids are small, such as the non-occurrence time and the initial occurrence time of the honeysuckle aphids, and can be used for controlling the aphids for a long time. After the target leaves are collected and applied with the pesticide, the content of 4 effective components in the leaves is reduced after the aphid is damaged, and the content of the four components gradually recovers to the normal leaf level along with the increase of the pesticide application time after the pesticide carrying microspheres are sprayed. This also describes β -caryophyllene matrine @ NH2The HMSN can slowly release the pesticide effect, effectively prevent and treat honeysuckle aphids and restore the damage of the aphids to target leaves.
(3) The wax layer of the leaf contains a large amount of lipophilic compounds and carboxyl, aldehyde and glycosidic bond groups, so that the leaf has hydrophobicity and negative charge, and beta-caryophyllene-matrine @ NH2-NH modified on surface of HMSN drug-carrying microsphere2In solutionHas positive charge, can be in hydrogen bond with-OH in the wax layer, can be in electrostatic adsorption with-COOH and can be in covalent bonding with-CHO. A series of complex interaction mechanisms can enhance the affinity of the drug-loaded microspheres on the target leaf surface and improve the wettability and retention capacity of the drug-loaded microspheres on the target leaf surface.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
Fig. 1 is a flow chart of a preparation method of the pesticide compound drug-loaded microsphere provided by the embodiment of the invention.
Fig. 2(a) is a mass spectrum of dodecane, an internal standard substance provided in the embodiment of the present invention.
FIG. 2(b) is a mass spectrum of dicyclohexyl phthalate provided in the example of the present invention.
Fig. 2(c) is a mass spectrum of a standard β -caryophyllene provided in the example of the present invention.
FIG. 2(d) is the mass spectrum of matrine provided in the example of the present invention.
FIG. 3(a) is a total gas mass ion flow diagram of a hybrid standard provided by an embodiment of the present invention;
in the figure: 1. dodecane; 2. beta-caryophyllene; 3. matrine; 4. dicyclohexyl phthalate.
FIG. 3(b) is a schematic diagram of β -caryophyllene matrine @ NH provided in an embodiment of the present invention2-gas mass total ion flow diagram of HMSN microspheres;
in the figure: 1. dodecane; 2. beta-caryophyllene; 3. matrine; 4. dicyclohexyl phthalate.
FIG. 4(a) is an HPLC chromatogram of a mixed control solution provided by an embodiment of the present invention;
in the figure: 1. chlorogenic acid; 2. luteolin; 3. isochlorogenic acid A; 4. isochlorogenic acid C.
FIG. 4(b) is an HPLC chromatogram of a test solution provided in an embodiment of the present invention;
in the figure: 1. chlorogenic acid; 2. luteolin; 3. isochlorogenic acid A; 4. isochlorogenic acid C.
FIG. 5 is a schematic diagram of honeysuckle cultivated in a greenhouse according to an embodiment of the present invention.
Fig. 6(a) is a scanning electron microscope image of an MSN according to an embodiment of the present invention.
Fig. 6(b) is a scanning electron micrograph of an HMSN according to an embodiment of the present invention.
Fig. 7(a) is a transmission electron microscope image of an MSN according to an embodiment of the present invention.
Fig. 7(b) is a transmission electron micrograph of HMSN according to an embodiment of the present invention.
FIG. 8 is an SEM image of silica microspheres at various CTAB dosages as provided by an example of the present invention.
FIG. 8(a) is an SEM image of silica microspheres with a large magnification of 14.57KX and CTAB in an amount of 0.15mL provided by an example of the invention.
FIG. 8(b) is an SEM image of silica microspheres with a magnification of 11.35KX and CTAB in an amount of 0.3mL provided by an example of the invention.
FIG. 8(c) is an SEM image of silica microspheres with a magnification of 13.69KX and CTAB in an amount of 0.4mL provided by an example of the invention.
FIG. 8(d) is an SEM image of silica microspheres with a magnification of 15.71KX and CTAB in an amount of 0.6mL provided by an example of the invention.
FIG. 9 is an SEM image of silica microspheres with different amounts of ammonia water according to embodiments of the present invention.
FIG. 9(a) is an SEM image of silica microspheres with a magnification of 14.76KX and an amount of 1mL ammonia provided by an example of the present invention.
FIG. 9(b) is an SEM image of silica microspheres with a magnification of 11.59KX and an ammonia dosage of 2mL according to an embodiment of the invention.
FIG. 9(c) is an SEM image of silica microspheres of the invention with a magnification of 13.87KX and an amount of 3mL ammonia water.
FIG. 9(d) is an SEM image of silica microspheres with a magnification of 15.35KX and an amount of ammonia of 4mL according to an embodiment of the present invention.
FIG. 10 is an SEM image of silica microspheres at various temperatures provided by an example of the present invention.
FIG. 10(a) is an SEM image of silica microspheres of the present invention provided in example with a magnification of 2.00KX and a reaction system temperature of room temperature.
FIG. 10(b) is an SEM image of silica microspheres of 2.34KX magnification and reaction system temperature of 35 ℃ provided by example of the present invention.
FIG. 10(c) is an SEM image of silica microspheres with a magnification of 5.00KX and a reaction system temperature of 80 ℃ provided in example of the present invention.
FIG. 11 shows MSN and NH provided by an embodiment of the present invention2-HMSN and beta-caryophyllene matrine @ NH2-fourier infrared spectrogram of HMSN.
FIG. 12 shows NH provided by an embodiment of the present invention2-HMSN and beta-caryophyllene matrine @ NH2-X-ray diffraction pattern of HMSN.
Fig. 13 is a schematic view of drug loading rates of drug loaded microspheres at different drug loading ratios according to an embodiment of the present invention.
FIG. 14 shows NH provided by an embodiment of the present invention2-HMSN, beta-caryophyllene matrine @ NH2Thermogravimetric analysis of physical mixture of HMSN, microspheres and pharmaceutical agents.
FIG. 15 shows a beta-caryophyllene matrine complex drug, a silica dioxide microsphere, and a beta-caryophyllene matrine @ NH provided in an embodiment of the present invention2-release profile of HMSN.
FIG. 16 is a graph showing the content changes of 4 effective components in target leaves before and after administration according to the embodiment of the present invention.
Fig. 17 is a contact angle image of different agents on honeysuckle flower foliage according to an embodiment of the invention.
FIG. 18 shows β -caryophyllene matrine @ NH provided in an embodiment of the present invention2SEM pictures of HMSN on honeysuckle leaves.
FIG. 18(a) is a schematic diagram of [ beta ] -caryophyllene, matrine @ NH provided by an embodiment of the present invention2S of HMSN on honeysuckle leaves before Water scouringEM image.
FIG. 18(b) is a schematic diagram of β -caryophyllene matrine @ NH provided in an embodiment of the present invention2SEM picture of HMSN on honeysuckle leaves after water-rinsing.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides an insecticide compound drug-loaded microsphere for honeysuckle and a preparation method thereof, and the invention is described in detail with reference to the accompanying drawings.
The compound synergistic composition provided by the embodiment of the invention is any one of thymol, beta-caryophyllene and matrine or trans-cinnamaldehyde and azadirachtin.
The mass ratio of thymol to beta-caryophyllene provided by the embodiment of the invention is 0.75:1-12:1, the mass ratio of beta-caryophyllene to matrine is 1:1-16:1, and the mass ratio of trans-cinnamaldehyde to azadirachtin is 0.45:1-4.2: 1.
The mass ratio of thymol beta-caryophyllene provided by the embodiment of the invention is 3:4, 9:7, 2:1, 3:1, 9:2, 7:1, 12:1 or 2.2: 1; the mass ratio of the beta-caryophyllene and the matrine is 1:1, 12:7, 8:3, 4:1, 6:1, 28:3, 16:1 or 2.4: 1; the mass ratio of the trans-cinnamaldehyde to the azadirachtin is 9:20, 27:35, 6:5, 9:5, 27:10, 21:5 or 1.6: 1.
The thymol beta-caryophyllene compound provided by the embodiment of the invention is a thymol beta-caryophyllene compound agent with the mass percentage of 16%, wherein the thymol mass percentage is 11%, and the beta-caryophyllene mass percentage is 5%; the beta-caryophyllene and matrine compound agent is 17% by mass, the beta-caryophyllene and matrine compound agent is 12% by mass and the matrine compound agent is 5% by mass; the trans-cinnamic aldehyde-azadirachtin compound is 13% by mass of the trans-cinnamic aldehyde-azadirachtin compound, the trans-cinnamic aldehyde is 8% by mass of the trans-cinnamic aldehyde-azadirachtin compound, and the azadirachtin is 5% by mass of the trans-cinnamic aldehyde-azadirachtin compound. As shown in fig. 1, the preparation method of the pesticide compound drug-loaded microsphere provided by the embodiment of the invention comprises the following steps:
s101, preparing hollow silica microspheres HMSN;
s102, amino-modified hollow silica microsphere NH2-preparation of HMSN;
s103, carrying out beta-caryophyllene-matrine @ NH2Preparation of HMSN drug-loaded microspheres.
The technical solution of the present invention will be further described with reference to the following examples.
Summary of the invention
The invention researches the influence of 21 plant source substances on the biological activity of honeysuckle aphids and screens the plant source substances with higher aphid killing activity; carrying out compounding research on the screened plant source substances, and determining the combination of the compound agents with synergistic action; the slow-release microspheres are prepared by loading a compound preparation on hollow silica microspheres, and a synergistic mechanism of the slow-release microspheres in the aspect of controlling honeysuckle aphids is discussed.
The method adopts a capillary dripping and disc method to determine the contact poisoning and stomach poisoning activity of 21 botanical substances on honeysuckle aphids; screening the optimal compound agent and the mixture ratio by using a co-toxicity factor and co-toxicity coefficient method, determining the toxicity of the compound agent by using an insect-soaking leaf-soaking method, judging the synergistic effect of the compound agent, and evaluating the effect of the compound agent on controlling honeysuckle aphids through pot culture and field experiments; synthesizing hollow silicon dioxide microspheres by adopting a soft template method, and preparing a sustained-release drug delivery system by using the hollow silicon dioxide microspheres as a carrier; carrying out related characterization and sustained-release performance analysis on the microspheres by using instruments such as a transmission electron microscope and a gas chromatograph-mass spectrometer, and analyzing the effect of the drug-loaded microspheres on controlling honeysuckle aphids through a pharmacodynamic test and target leaf component change; the wettability and the retention of the drug-loaded microspheres on the target blades are measured by using a contact angle measuring instrument and a scanning electron microscope, so that the synergistic mechanism of the drug-loaded microspheres for preventing and treating honeysuckle aphids is determined.
Experiments of the invention show that 21 plant source substances have different degrees of contact poisoning and stomach poisoning activities on honeysuckle aphids, and 5 plant source substances with different degrees of contact poisoning and stomach poisoning activities are obtained by analyzing the corrected death rates of 24, 48 and 72 hours of aphids after treatmentPlant source substances with strong aphid killing activity: thymol, beta-caryophyllene, trans-cinnamaldehyde, matrine and azadirachtin. 2. The compound research of 5 plant source substances is carried out, and 3 compound combinations with obvious synergistic effect are as follows: 16% thymol beta-caryophyllene, 17% beta-caryophyllene matrine and 13% trans-cinnamaldehyde azadirachtin. The toxicity test result shows that the toxicity of the 3 compound agents on honeysuckle aphids is enhanced, and the 17 percent beta-caryophyllene matrine and the 13 percent trans-cinnamaldehyde azadirachtin have obvious synergistic effect on respective raw pesticide. The field test shows that the effect of preventing and treating honeysuckle aphid is optimal by 17% of beta-caryophyllene matrine, and the prevention and treatment effect is about 61% and 91% after the pesticide is applied for 1d and 10 d. 3. The experiment shows that the beta-caryophyllene matrine @ NH is obtained2The optimal preparation conditions for HMSN drug delivery systems are as follows: dissolving 0.3000g CTAB in the mixed solution (60mL of absolute ethyl alcohol and 100mL of deionized water), magnetically stirring, adding 2mL of concentrated ammonia water, dropwise adding 2mL of TEOS, and reacting at room temperature for 6 h; centrifuging to obtain white precipitate, expanding pores with cyclohexane, calcining in a muffle furnace and modifying with amino to obtain NH2-HMSN as β -caryophyllene matrine: NH (NH)2-HMSN mass ratio of 1:1 preparing drug-loaded microspheres. The result shows that the drug loading rate under the condition is about 36.13 percent, the cumulative release rate after 288 hours is more than 80 percent, and the slow release effect is obvious; the pharmacodynamic test shows that the beta-caryophyllene matrine @ NH2HMSN not only has good effect of preventing and treating honeysuckle aphids and quick effect, but also can slowly release the medicament to play a role in prolonging the pesticide effect time, the prevention and treatment effect can reach 96% after 15 days of pesticide application, and the damage of the aphids on the effective components of honeysuckle leaves can be repaired; the research on the synergism discovers that the beta-caryophyllene matrine @ NH2The HMSN can enhance the affinity of the liquid medicine and the target leaf surface by improving the wettability and the deposition performance of the liquid medicine on the target leaf surface.
Therefore, the 21 botanical substances have contact poisoning and stomach poisoning effects of different degrees on the honeysuckle aphids, and the botanical substances with high activity and wide effect are screened out in experiments for compounding, so that the aphid killing activity of the original pesticide is enhanced. The hollow silica microsphere loaded compound preparation is used for preparing the slow-release drug-carrying microsphere, so that active ingredients of liquid medicine can be effectively protected, the quick-acting property, the slow-release property and the lasting effect of preventing and treating honeysuckle aphids are improved, the drug release can be matched with the aphid occurrence rule, the effect of inhibiting the population growth of the aphids is achieved, and an important direction is provided for the research of safe, green and efficient prevention and treatment of the honeysuckle aphids.
According to the invention, honeysuckle aphids are used as test insects, honeysuckle is used as an experimental material, plant source substances with high activity and wide effect are screened, the aphid killing activity of a single dose is enhanced through compounding, and the slow-release drug-carrying microspheres are prepared, so that the quick action and high efficiency of the plant source liquid medicine for preventing and controlling the aphids can be improved, the lasting period is prolonged, and the harm of the aphids to the honeysuckle is reduced. The invention lays a foundation for green and efficient control of honeysuckle aphids, provides a reference way for reducing pesticide application and increasing pesticide efficiency, and has important significance for high-quality and efficient cultivation of Chinese medicinal materials.
Secondly, 21 influence of plant source substances on biological activity of honeysuckle aphids
With the increasingly prominent use of chemical pesticides, green pesticides with the advantages of greenness, high efficiency, lasting effect, aftereffect and the like become hot contents for research. Plant-derived substances are widely concerned in the field of pest control because of their advantages of safety, low toxicity, wide sources, various mechanisms of action, and the like. Early experiments show that thyme oil, oregano oil and trans-2-hexenal have strong activity on honeysuckle aphids, and 21 plant source substances are determined for experimental research by analyzing thyme oil, oregano oil and active ingredients of homologous plants and reading related documents. The invention determines the contact poisoning and stomach poisoning activities of 21 botanical substances on honeysuckle aphids, and screens out the botanical substances with stronger aphid killing activity.
1. Test material
The aphids of honeysuckle and tender tips of honeysuckle are all from the medicine garden of Shandong Chinese medicine university.
2. Instruments and reagents
2.1 instruments
A multifunctional constant temperature incubator (Beijing Fujiu) and a high temperature sterilization box (MJ-78A, Kyowa Kai, Shanghai).
2.2 reagents
The experimental reagents are detailed in table 1.
TABLE 1 test reagents
3. Experimental methods
3.121 influence of plant source substance on biological activity of honeysuckle aphid
Selecting wingless adult aphids with consistent sizes, dissolving the plant source substances with acetone, and preparing the experiment liquid medicine with a 0.1% Tween-80 aqueous solution.
3.1.1 contact killing Activity assay
According to a capillary dripping method, honeysuckle aphids are used as test insects, 20 aphids are collected in each group, 0.1ul of experiment liquid medicine is dripped in each group, each treatment is repeated for 3 times, dripping acetone is used as a solvent control, dripping clear water is used as a blank control, and the experiment is abandoned if the mortality rate of the control is higher than 20%. After treatment, aphids are picked into a culture dish with moisturized honeysuckle leaves, the fresh leaves are replaced every 24 hours, the culture dish is placed in a constant temperature box with the temperature of (25 +/-1) DEG C and the relative humidity of (60 +/-5)% for continuous culture, the survival conditions of the aphids are checked respectively 24, 48 and 72 hours after treatment, and the corrected death rate is calculated.
3.1.2 stomach toxicity Activity assay
Reference to disc method for determination of biological activity of aphids. The 2% agar solution was sterilized at high temperature, poured into a petri dish (d 9cm) and spread with a moisture-retaining layer of about 0.5cm thick. Soaking the leaves of flos Lonicerae in the medicinal liquid for 5s, drying the residual medicinal liquid with filter paper, naturally drying, sticking the front of the leaves on agar, and inserting the petiole into the agar. Picking (20 +/-5) aphids on the leaves, sealing the culture dish with a sealing film to prevent the aphids from escaping, pricking holes with insects to ensure air permeability, and continuously culturing in a constant temperature box with the temperature of (25 +/-1) DEG C and the relative humidity of (60 +/-5)%. Acetone is used as a medicament control, clear water treatment is used as a blank control, each treatment is repeated for 3 times, aphid survival conditions are checked after 24 hours, 48 hours and 72 hours respectively, and the corrected mortality rate is calculated.
3.2 indoor toxicity determination of honeysuckle aphids by different botanical substances
Cutting tender tips of honeysuckle with aphids by adopting an insect-soaking and leaf-soaking method recommended by FAO, wrapping cut tips with wet absorbent cotton and a preservative film for moisture preservation, and keeping (40 +/-5) heads of wingless adult aphids on each tender tip. Soaking tender tips of honeysuckle with aphids in the liquid medicine for 5s, sucking the redundant liquid medicine with filter paper, transferring the liquid medicine to a culture dish paved with wet filter paper and with the diameter of 9cm, drying the liquid medicine, sealing with a sealing film to prevent the aphids from escaping, puncturing holes with insects, and ensuring the air permeability of the culture dish. The culture was then continued in an incubator at a temperature of (25. + -. 1 ℃ C.) and a relative humidity of (60. + -. 5)% and each treatment was repeated 3 times. Aphid survival was checked after 48h and LC of different plant-derived substances was calculated using SPSS 2550。
4. Results and analysis
4.121 contact killing activity of plant source substance on honeysuckle aphid
As can be seen from Table 2, the contact killing activity of beta-caryophyllene on honeysuckle aphids is strongest, the death rate after 24 hours is more than 90%, and the death rate after 48 hours is about 98%. 5 plant-derived substances with the corrected mortality rate of more than 70 percent in 24 hours are respectively beta-caryophyllene, trans-cinnamaldehyde, thymol, matrine and azadirachtin, and 4 plant-derived substances with the corrected mortality rate of 50 to 70 percent are respectively carvacrol, alpha-pinene, citronellal and nicotine; the plant-derived substances with corrected mortality rate of more than 80% after 72 hr comprise thymol, carvacrol, beta-caryophyllene, trans-cinnamic aldehyde, matrine, and azadirachtin. In conclusion, the contact activity of the beta-caryophyllene and the trans-cinnamaldehyde is high, and the insecticidal speed is high.
TABLE 221 contact killing activity of plant-derived substances on honeysuckle aphids
Note: the data in the table are the average of 3 replicates. The concentration of nicotine, matrine and azadirachtin liquid medicine is 300 mg/L; the concentration of the other plant source substance liquid medicine is 1000 mg/L. The same applies below.
4.221 stomach toxicity activity of plant source substance to honeysuckle aphid
According to the stomach toxicity activity result, the plant-derived substances with the death rate of more than 50 percent corrected for 24 hours comprise thymol, beta-caryophyllene, matrine and azadirachtin; 6 plant-derived substances with death rate of more than 70% after 72h correction are thymol, beta-caryophyllene, trans-cinnamaldehyde, matrine, nicotine and azadirachtin respectively. In conclusion, the matrine has good stomach toxicity activity on honeysuckle aphids, but the insecticidal speed and the insecticidal intensity are to be improved. As can be seen by comparing the results of the contact-killing and stomach-poisoning activities of 21 plant-derived substances, the contact-killing activity of 21 plant-derived substances on honeysuckle aphids is slightly higher than the stomach-poisoning activity overall (see Table 3).
Stomach toxicity activity of plant source substances in Table 321 on honeysuckle aphid
Combining the results of the 21 plant source substance contact activity and stomach toxicity activity determination, and taking the contact activity corrected mortality rate of more than 80% and the stomach toxicity activity corrected mortality rate of more than 70% as the screening standard to obtain 5 plant source substances with stronger aphid killing activity, which are respectively beta-caryophyllene, trans-cinnamaldehyde, thymol, matrine and azadirachtin.
4.35 indoor toxicity analysis of plant-derived substances
According to the results of the indoor toxicity test of 5 plant source substances, the toxicity of the matrine to honeysuckle aphids is the highest, and LC5026.859mg/L, and the toxicity of the other 4 plant source substances is from high to low: nimbinLC50Is 90.377mg/L, beta-caryophyllene LC50108.004mg/L, trans-cinnamaldehyde LC50162.081mg/L of thymol LC50It was 303.833mg/L (see Table 4).
TABLE 45 determination of toxicity of plant-derived substances on honeysuckle aphids
5. Discussion and summary
The action of the plant source substance on pests is mainly shown as poisoning, avoiding, attracting and the like, and the invention researches the poisoning action (namely contact poisoning and stomach poisoning) of 21 plant source substances and obtains the following conclusion:
the 21 botanical substances have stronger contact killing activity on honeysuckle aphids, and the analysis reason may be that the aphids suck juice through a piercing-sucking mouthpart for feeding instead of chewing for feeding, the stomach toxicity of the liquid medicine is difficult to play through the alimentary canal or the trachea, and the liquid medicine is in various modes of directly contacting the aphids, so the liquid medicine can play a role more easily. And (3) analyzing results of 24, 48 and 72 hours after the liquid medicine is treated, and screening 5 plant source substances with high activity and taking contact killing and stomach poisoning effects into consideration: thymol, beta-caryophyllene, trans-cinnamaldehyde, matrine and azadirachtin. The toxicity of 5 plant source substances is measured by adopting an impregnation method recommended by FAO, and the toxicity of each plant source substance is obtained, wherein the toxicity is matrine, azadirachtin, beta-caryophyllene, trans-cinnamaldehyde and thymol.
Compatibility test between different plant source substances
Botanical pesticides play an important role in pest control, but are limited by the disadvantages of low efficacy, short duration of action, slow efficacy, and the like. In recent years, researchers find in the aspect of compounding and synergism that the reasonable compatibility of various components with insecticidal activity can obviously improve the insecticidal activity of original drugs and enhance the effect, such as chrysanthemum, matrine soluble solution, avermectin, azadirachtin emulsion and the like. Based on the above, the formulation research is carried out on 5 plant-derived substances with higher aphid killing activity screened in the earlier stage, the formulation combination is qualitatively screened by using a co-toxicity factor method, the screened formulation combination is quantitatively analyzed by using a co-toxicity coefficient method on the basis, and the formulation combination for efficiently preventing and treating honeysuckle aphids is obtained through synergy and efficacy evaluation.
1. Instruments and reagents
Thymol, beta-caryophyllene, trans-cinnamaldehyde, matrine and azadirachtin refer to '21 plant source substances influencing biological activity of honeysuckle aphids' medium '2.2 reagent'; 10% imidacloprid emulsifiable concentrate purchased from Qingdao Zhengdao pharmaceutical Co.
2. Experimental methods
2.1 screening of synergistic combinations of botanical substances
The method referred to Mansour et al is modified and specifically designed as follows: LC for honeysuckle aphid with single medicament50Values are benchmark, assuming LC of virulence determination agent A, B50Respectively a and b, selecting the sextant points of the addition action line in the equivalent line method as 5 mixture ratios, representing as a/5b, a/2b, a/b, 2a/b and 5a/b, calculating the co-toxicity factors, and screening the compound combination with the synergistic action.
The synergistic effect is obtained when the co-toxicity factor is more than or equal to 20, the antagonistic effect is obtained when the co-toxicity factor is less than or equal to-20, and the additive effect is obtained when the co-toxicity factor is more than-20 and less than-20.
2.2 determination of optimum compounding ratio of compounded composition
According to the 2.1 experimental result, the optimum proportion of the compound combination is screened out by referring to a Sunpivoton cotoxicity coefficient (CTC) method and a pesticide compound optimum synergistic formula screening method, 9 concentration proportions are set at the ten equi-division points of the addition action line in the equivalent line method, and can be expressed as a/9b, a/4b, 3a/7b, 2a/3b, a/b, 3a/2b, 7a/3b, 4a/b and 9a/b, according to the proportion of 1: 9. 1: 4. 3: 7. 2: 3. 1: 1. 3: 2. 7: 3. 4: 1. 9: 1 to obtain the concentration gradient of the compound agent. And screening out the optimal mixture ratio of the compounded combination by combining the co-toxicity coefficient with an SPPSS mathematical model.
The antagonism is when the CTC is less than 80, the addition action is when the CTC is less than 80, the synergism is when the CTC is more than 120, and the remarkable synergism is realized when the CTC is more than 200.
2.3 indoor toxicity determination of Compound agent on honeysuckle aphid
Referring to a method for determining indoor toxicity of 3.2 different plant source substances to honeysuckle aphids, 10% imidacloprid EC is used as a control medicament, clear water is used as a blank control, and LC of the compound agent is determined50And analyzing whether the compound agent has the synergistic effect on each original medicine or not by taking the non-overlapping 95% confidence intervals as the judgment standard of whether the obvious synergistic effect exists before and after the compound.
2.4 test of the efficacy of the combination agent
2.4.1 potting test
The prevention and control effect is observed through a potted plant spray test, and the method comprises the following specific steps: when the potted honeysuckle grows to 6-10 leaves, inoculating the aphid (40 +/-5) head of the honeysuckle to each plant, covering an insect-proof net, investigating the insect population after 24 hours, and applying the pesticide by using a small handheld sprayer after the insect population is stable. The treatment groups are respectively applied with 500 times of liquid, 750 times of liquid and 1000 times of liquid, the medicament is applied according to the field recommended concentration by contrasting with 10 percent imidacloprid EC, and clear water is blank contrast. 1 pot of honeysuckle is treated each time and is repeated for 3 times, the number of the survived aphids is recorded at 1, 3, 7, 10 and 15 days after the application, and the control effect is calculated and corrected according to the insect population reduction rate.
2.4.2 field test
Spraying the pesticide on the ground in a Shandong Chinese medicinal university medicine garden in 2019, 4 and 2 months, wherein the weather is clear on the pesticide application day. Dividing the land mass for pesticide application into different cells with the area of 15m2A five-point sampling method is adopted, 10 honeysuckle flowers are selected in each cell, branches with the length of 15cm and the consistent growth vigor are randomly selected in east, south, west, north and middle 5 directions of each honeysuckle flower, and the branches are marked by hanging labels. And 1m protection lines are arranged around the test area and among cells to prevent interference. The manual sprayer is adopted for applying the pesticide, and the spray head is inclined to the lower side surface of the plant, so that the front and back surfaces of the blade are uniformly sprayed with the pesticide, but the pesticide liquid does not drip. And selecting the concentration of the compound agent in the field test according to the potted plant test result, taking clear water as a blank control and 10% imidacloprid EC missible oil as a medicament control, repeating each treatment for 3 times, recording the number of the surviving aphids after the application for 0, 1, 4, 7, 10 and 14d, and calculating the correction control effect by the same method.
3. Results and analysis
3.1 screening results of synergistic combinations
According to the determination result of the co-toxicity factor, the thymol beta-caryophyllene is compounded in the ratio of 28: the composition ratio of 5 is 21.18, the co-toxicity factor has synergistic effect, and the compounding of thymol and other 3 plant source substances is mostly additive effect. The beta-caryophyllene and matrine are compounded in a proportion of 4: the composition ratio of 5 is more than 20, the co-toxicity factor has synergistic effect, and the beta-caryophyllene and other 2 plant source substances are compounded to have additive effect. The trans-cinnamaldehyde and azadirachtin are compounded in a weight ratio of 9: 10 and 9: the 5 proportion has synergistic effect, the combined poisoning factors of the trans-cinnamic aldehyde and the matrine are all less than 20, and no synergistic effect exists (see table 5).
TABLE 5 determination results of mutual toxicity factors of thymol and thymol combination on honeysuckle aphid
Note: the preparation concentrations of thymol, caryophyllene, trans-cinnamaldehyde, matrine, and azadirachtin are 300, 108, 180, 30, and 90mg/L respectively. The same applies below.
3.2 determination of optimum mixture ratio of Compound combination
3.2.1 optimal proportioning of thymol beta-caryophyllene
According to the result of the co-toxicity coefficient measurement, the ratio of thymol to beta-caryophyllene is 3: 4. 9: 7. 2: 1. 3: 1. 9: 2. 7:1 and 12: when 1, the actual co-toxicity coefficient is more than 120, and the synergistic effect is achieved. Fitting the relationship between the co-toxicity coefficient Y and the beta-caryophyllene percentage content arcsine conversion value X by using SPSS to obtain a unitary quadratic mathematical model Y which is-0.169X2+11.410X +17.650, the test results show that the equation is representative (see Table 6-2). The theoretical maximum cotoxicity coefficient is 157.60, the percentage content of beta-caryophyllene is 30.80%, and the ratio of thymol to beta-caryophyllene is (1-30.80%): 30.80% ≈ 2.2:1 is the best proportioning.
TABLE 6-1 determination of co-toxicity coefficient of thymol and beta-caryophyllene at different ratios to aphid in honeysuckle
TABLE 6-2 analysis of variance table of quadratic regression
Note: looking up table to know F (2,6)0.05＝5.14；F(2,6)0.0110.92. The same applies below.
3.2.2 optimal proportion of beta-caryophyllene and matrine
According to the measurement result, the proportion of the beta-caryophyllene and the matrine is 1: 1. 12: 7. 8: 3. 4: 1.6:128:3 and 16:1, it has synergistic effect. Fitting the relationship between co-toxicity coefficient y and matrine percentage content arcsine conversion value X with SPSS data processing software to obtain a unitary quadratic mathematical model y of-0.221X2+14.545X-35.544, the test results show that the equation is representative (see Table 7-2). The theoretical maximum cotoxicity coefficient is 203.77, the percentage content of matrine is 29.52%, and the ratio of matrine to beta-caryophyllene is 29.52% according to the equation: (1-29.52%). apprxeq.1: 2.4 is the best proportioning.
TABLE 7-1. beta. -caryophyllene and matrine different mixture ratio determination result of co-toxicity coefficient of honeysuckle aphid
TABLE 7-2 analysis of variance of quadratic regression
3.2.3 optimum ratio of trans-cinnamic aldehyde-azadirachtin
From the measurement results, the ratio of trans-cinnamaldehyde to azadirachtin is 9: 20. 27: 35. 6: 5. 9: 5. 27:10 and 21:5, it has synergistic effect. Fitting the relationship between the co-toxicity coefficient y and the azadirachtin percentage content arcsine conversion value X by using SPSS data processing software to obtain a unitary quadratic mathematical model y which is-0.132X2+10.216X-14.768, the test results show that the equation is representative (see Table 8-2). The theoretical maximum cotoxicity coefficient is 182.90, the percentage content of nimbin is 39.09%, and the ratio of nimbin and trans-cinnamaldehyde is 39.09% according to the equation: (1-39.09%). apprxeq.1: the optimal ratio is 1.6.
TABLE 8-1 determination of co-toxicity coefficient of different proportions of trans-cinnamaldehyde and azadirachtin to honeysuckle aphid
TABLE 8-2 analysis of variance of quadratic regression
The following results were obtained by compounding experiments: when the ratio of thymol beta-caryophyllene is 2.2: 1. the proportion of beta-caryophyllene and matrine is 2.4: 1. the proportion of trans-cinnamic aldehyde to nimbin is 1.6:1, 3 compound preparations are respectively prepared into a thymol beta-caryophyllene compound agent with the content of 16% (wherein the thymol content is 11% and the beta-caryophyllene content is 5%), a beta-caryophyllene matrine compound agent with the content of 17% (wherein the beta-caryophyllene content is 12% and the matrine content is 5%), a trans-cinnamaldehyde azadirachtin compound agent with the content of 13% (wherein the trans-cinnamaldehyde content is 8% and the azadirachtin content is 5%).
3.3 virulence analysis of prevention and treatment of honeysuckle aphids by the combination
As can be seen from Table 9, the toxicity of 17% beta-caryophyllene matrine was the highest among the 3 combinations, and LC5013.997mg/L, followed by 13% trans-cinnamaldehyde azadirachtin and 16% thymol beta-caryophyllene, LC5061.098mg/L and 91.093mg/L respectively, and the three compound agents have enhanced toxicity compared with the original drug. Comparing the results in table 5, the beta-caryophyllene-matrine compound agent and the trans-cinnamaldehyde-azadirachtin compound agent have obvious synergistic effect on the original medicine, and the thymol beta-caryophyllene only has obvious synergistic effect on the thymol.
TABLE 93 toxicity test results of the combination agent on aphids of honeysuckle
3.4 potting test
As can be seen from tables 10-1 and 10-2, the quick action of preventing and treating honeysuckle aphids is improved after the single dose is compounded, and the aphid killing activity of the pesticide is enhanced. Compared with thymol and beta-caryophyllene single agents, the 16% thymol beta-caryophyllene compound agent has the effect of preventing and controlling honeysuckle aphids which are respectively improved by 7% -14% and 2% -3%; compared with the single dosage of beta-caryophyllene and matrine, the effect of the 17 percent beta-caryophyllene and matrine compound agent for preventing and controlling honeysuckle aphids is respectively improved by 13 to 19 percent and 6 to 15 percent; compared with a single dose of trans-cinnamaldehyde and azadirachtin, the 13 percent of the compound dose of trans-cinnamaldehyde and azadirachtin has the effect of preventing and controlling honeysuckle aphids which is respectively improved by 10 percent to 16 percent and 3 percent to 9 percent. The control effect of the 3 compound preparations is gradually increased after the compound preparations are applied for 1-10 days, then the control effect begins to be reduced, the control effect is better after the compound preparations are diluted by 300 times of 16% thymol, beta-caryophyllene and matrine, 300 and 500 times of 17% beta-caryophyllene and matrine, and 300 times of 13% trans-cinnamaldehyde and azadirachtin, the control effect is 83.94%, 92.33%, 91.24% and 87.58% respectively after the compound preparations are applied for 10 days, wherein the control effect of the 17% beta-caryophyllene and matrine compound preparation is not different from that of imidacloprid, a chemical pesticide.
In conclusion, a field test was carried out by selecting a 300-time solution of a 16% thymol-beta-caryophyllene compound, a 500-time solution of a 17% beta-caryophyllene-matrine compound, and a 300-time solution of a 13% trans-cinnamic aldehyde-azadirachtin compound.
Table 10-15 single-dose potting test for controlling honeysuckle aphids
Note: data in the table are mean ± sem. Different letters after the same column of data indicate significant differences at P < 0.05 levels by Duncan's New Complex Pole Difference method. The same applies below.
Table 10-23 potting experiment for controlling honeysuckle aphids by using compound agent
3.5 field test
From Table 11, the quick action and the effect of the plant source substances for preventing and treating honeysuckle aphids are improved after the plant source substances are compounded, wherein the prevention and treatment effect is the best by 17 percent of the beta-caryophyllene and matrine compound agent, the prevention and treatment effect is about 61 percent after 1d of application, the prevention and treatment effect reaches 91 percent after 10d of application, and the prevention and treatment effect is obviously higher than that of other two compound agents (P is less than 0.05); 16% thymol beta-caryophyllene has the worst quick-acting property and low effect, and cannot effectively prevent and control aphids. Compared with 10% imidacloprid EC, the quick-acting property and high efficiency of the 17% beta-caryophyllene and matrine compound agent need to be enhanced.
Table 113 compound agent for field control effect on honeysuckle aphids
4. Discussion and summary
The botanical pesticide has long application history, has the advantages of safety, low toxicity, various effects and the like, but is difficult to meet the requirement of controlling pests due to the defects of single dosage form, slow effect, short effect and the like. Based on this, the present chapter combines the judgment method of the predecessor for the pesticide compound synergistic screening to carry out compound synergistic research on 5 medicaments, and the results are as follows:
firstly, 9 compound combinations of 5 medicaments are qualitatively screened by utilizing a co-toxicity factor to obtain 3 compound combinations with synergistic action, namely thymol, beta-caryophyllene, matrine, trans-cinnamaldehyde and azadirachtin; then, the co-toxicity coefficient is combined with mathematical fitting model analysis to carry out quantitative analysis on the 3 compound combinations, and the optimal mixture ratio of the compound combinations is thymol, beta-caryophyllene (the mixture ratio is 2.2: 1), beta-caryophyllene, matrine (the mixture ratio is 2.4: 1) and trans-cinnamaldehyde, azadirachtin (the mixture ratio is 1.6: 1). Based on this, 16% thymol β -caryophyllene complex agent (thymol content 11%, β -caryophyllene content 5%), 17% β -caryophyllene and matrine complex agent (β -caryophyllene content 12%, matrine content 5%), and 13% trans-cinnamaldehyde and azadirachtin complex agent (trans-cinnamaldehyde content 8%, azadirachtin content 5%) were prepared, respectively.
The toxicity test result shows that: the 3 compound agents have enhanced toxicity to honeysuckle aphids, 17 percent of beta-caryophyllene and matrine have the strongest toxicity, and LC5013.997 mg/L; 17% of beta-caryophyllene matrine and 13% of trans-cinnamaldehyde azadirachtin have obvious synergistic effect on respective raw medicines, and 16% of thymol beta-caryophyllene has obvious synergistic effect on thymol raw medicines, but has synergistic or additive effect on beta-caryophyllene.
The efficacy test shows that the effect of preventing and controlling the aphids is enhanced and the quick action is improved after the single dose is compounded, wherein the effect of preventing and controlling the 17 percent beta-caryophyllene and matrine compound dose is optimal, which is related to the high toxicity of the compound dose, and the compound dose possibly has higher contact poisoning and stomach poisoning activities and better effect of preventing and controlling the honeysuckle aphids. The field test result and the potted plant test result are slightly different, the analysis reason is that the field pesticide application is greatly influenced by the environment such as temperature, illumination, wind power and the like, the loss of pesticide liquid is large after spraying, the pesticide liquid attached to the leaves is less, and the pesticide concentration is low after decomposition under high temperature and illumination, so the control effect is slightly lower than that of the potted plant test.
Synergism research of plant source substance compound agent
The prior commonly used pesticide preparation is mainly the traditional preparation forms such as missible oil, water aqua and the like, has the prominent problems of large organic solvent dosage, poor dispersibility, easy decomposition and the like, and has the defects of poor pesticide utilization rate, low pesticide effect, environmental pollution and the like. In recent years, researchers combine nano materials and technologies to synthesize effective carriers, and prepare a novel sustained-release preparation from a technical material with the defects of insolubility, easy decomposition, short duration and the like through ways of embedding or adsorption and the like, so that the dispersibility and stability of pesticide can be effectively improved, the adhesion and wettability of liquid medicine and target leaf surfaces are promoted, the liquid medicine and the target leaf surfaces are quickly spread to form a film, the duration of pesticide control is prolonged, the pesticide utilization rate is greatly improved, and the environmental pollution is reduced. Based on the above, the invention takes 17% beta-caryophyllene and matrine with the best prevention and treatment effect as the target, uses hollow silicon dioxide microspheres to prepare a new sustained-release microsphere formulation, and performs pesticide effect test and synergistic mechanism analysis so as to improve the quick action, high efficiency and lasting effect of the plant source compound agent for preventing and treating honeysuckle aphids in the field and provide a thought for developing a new green pesticide formulation.
1. Instruments and reagents
1.1 instruments
The experimental apparatus is detailed in table 12.
TABLE 12 Experimental apparatus
1.2 reagents
The reagents are detailed in Table 13.
TABLE 13 test reagents
2. Experimental methods
2.1 preparation and characterization of Slow Release drug-loaded microspheres
2.1.1 preparation of hollow silica microspheres (HMSN)
Reference is made to literature methods and modifications are made. Weighing a certain amount of hexadecyl trimethyl ammonium bromide, dissolving the hexadecyl trimethyl ammonium bromide in an ethanol solution (60mL of absolute ethanol and 100mL of deionized water), magnetically stirring, adding strong ammonia water, fully mixing, dropwise adding tetraethyl orthosilicate, and reacting for 6 hours at room temperature. Washing with deionized water and anhydrous ethanol for 3 times, centrifuging, dispersing the white precipitate into a mixed solution of 60mL of deionized water, 60mL of anhydrous ethanol and 9mL of cyclohexane, sealing, placing in an oven for 3 days at 60 ℃, filtering, washing with deionized water for 3 times, and freeze-drying. Calcining at 550 deg.C for 6 hr in muffle furnace, removing hexadecyl trimethyl ammonium bromide, and storing at room temperature under drying condition as HMSN. By changing reaction conditions, the influence of the TEOS/CTAB dosage, the ammonia water concentration and the temperature on the microsphere morphology is respectively researched.
2.1.1.1 influence of CTAB dosage on morphology of silica microspheres
The ammonia concentration and temperature were unchanged, the TEOS dose was 2mL, the CTAB doses were changed to 0.15, 0.3, 0.4, 0.6mL, respectively, and the morphology was observed under SEM.
2.1.1.2 influence of ammonia water dosage on morphology of silicon dioxide microspheres
The amount of CTAB and TEOS and the temperature were unchanged, 1, 2, 3 and 4mL of ammonia (28%) were added, and the morphology was observed under SEM.
2.1.1.3 Effect of temperature on morphology of silica microspheres
The CTAB and TEOS dosages and the ammonia water concentration are unchanged, the reaction temperature is respectively controlled to be room temperature, 35 ℃ and 80 ℃, and the morphology is observed under SEM.
2.1.2 amino-modified hollow silica microspheres (NH)2HMSN) preparation
According to the literature, 100mg of HMSN is taken and dispersed in absolute ethyl alcohol for 30min, a mixed solution containing 500 mu L of 3-aminopropyltriethoxysilane and 125 mu L of absolute ethyl alcohol is added into the dispersed solution, stirred for 24h at room temperature, centrifuged, washed and dried in vacuum to obtain white powder, which is marked as NH2-HMSN。
2.1.3 beta-caryophyllene matrine @ NH2Preparation of HMSN drug-loaded microspheres
Precisely weighing 100mg of beta-caryophyllene-matrine with content of 17%, adding appropriate amount of ethanol, ultrasonic treating to dissolve the medicine, preparing into medicinal liquid with mass concentration of 5mg/L, heating in water bath at 60 deg.C, mixing with NH2-HMSN mass ratio of 1: 2.2: 3. 1: 1. 2:1 addition of NH2And (4) carrying out ultrasonic treatment for 30min to fully disperse the components, stirring for 3h under a sealed condition, continuously stirring for 1h under an open condition, and volatilizing the organic solvent. Washing residual agent with hot ethanol, and freeze drying to obtain beta-caryophyllene-matrine @ NH with different proportions2-HMSN drug loaded microspheres.
2.1.4 related characterization of drug-loaded microspheres
2.1.4.1 topography Observation
Plating gold on MSN and HMSN, and observing the morphology under a Scanning Electron Microscope (SEM); adding MSN and HMSN into a proper amount of ethanol, performing ultrasonic dispersion, dripping a small amount of solution on a copper net with a carbon film, drying the copper net with the MSN and the HMSN under an infrared lamp, and then placing the copper net with the MSN and the HMSN under a Transmission Electron Microscope (TEM) to observe the appearance.
2.1.4.2 Fourier Infrared Spectroscopy
Uniformly grinding 1mg and 150mg of potassium bromide (KBr) of a sample, pressing the ground sample into a transparent sheet, drying the transparent sheet under an infrared lamp, and carrying out structural analysis, wherein the test range is 400-4000 cm-1Resolution 2cm-1The number of scans was 60.
2.1.4.3 particle size and potential measurements
The prepared beta-caryophyllene, matrine and NH2Diluting the HMSN drug-loaded microspheres to 0.1 mu g/mu L by using deionized water, sucking 1.5mL of sample solution, adding the sample solution into a cuvette, and measuring the hydrated particle size distribution and the Zeta potential condition by using a multi-angle particle size and high-sensitivity Zeta potential analyzer.
2.1.4.4X-ray diffraction (XRD) analysis
Investigation of NH Using copper target2-HMSN and beta-caryophyllene matrine @ NH2-crystal structure of HMSN drug loaded microspheres.
2.1.4.5 drug load determination
Precisely weighing 100mg of beta-caryophyllene-matrine @ NH prepared by different drug loading ratios2Putting HMSN drug-loaded microsphere powder into a 50mL conical flask with a plug, adding a proper amount of 50% ethanol solution, performing ultrasonic dispersion for 2 hours, taking a proper amount of supernatant, and determining the drug-loaded amount by GC-MS after treating.
2.1.4.6 Thermogravimetric (TG) analysis
0.2180g of beta-caryophyllene-matrine @ NH are weighed2HMSN at N2Raising the temperature from room temperature to 800 ℃ at a temperature raising rate of 10 ℃/min in the atmosphere, and analyzing by a thermogravimetric analyzer.
2.1.5 determination of sustained-release Properties of drug-loaded microspheres
50% absolute ethyl alcohol was used as release medium. Mixing 100mg of beta-caryophyllene, matrine and NH2HMSN was placed in dialysis bags, placed in centrifuge tubes containing 30mL of release medium, and the tubes were placed in a shaker at 25 ℃ with an oscillation rate of 200 r/min. After a certain time 2mL of solution are removed and the same volume of release medium is replenished. And (4) analyzing by adopting GC-MS (gas chromatography-Mass spectrometer), and calculating the accumulated release amount.
Where E is the cumulative release (%), V is the volume of release medium removed (2mL), Ci(mg/mL) is the concentration of drug at sample i, Cn(mg/mL) is the concentration of the drug at the time of sampling n, V0Volume of release medium (30mL), M (mg) is NH loading2-total mass of drug in HMSN.
2.1.5.1 chromatographic conditions
Agilent HP-5ms, 30m × 250 μm × 0.25 μm, FID detector; the column temperature is 100 ℃, the injection inlet temperature is 250 ℃, the flow rate is 1mL/min, the injection amount is 1 muL, and the split ratio is 20: 1. mass spectrum conditions: the ion source is EI, the temperature of the ion source is 230 ℃, the temperature of a quadrupole rod is 150 ℃, the electron energy is 70eV, the scanning mass range is 50-400 amu, and the solvent delay is 2 min. The temperature program is shown in Table 14.
TABLE 14 temperature programmed gradient
Preparation of 2.1.5.2 internal standard solution
Taking a proper amount of dodecane to be dissolved in a 100mL volumetric flask, and fixing the volume to a scale mark to prepare 8g/L internal standard solution 1.
Taking a proper amount of dicyclohexyl phthalate in a 100mL volumetric flask, dissolving with 50% ethanol, and fixing the volume to a scale mark to prepare an internal standard solution 2 of 4 g/L.
2.1.5.3 preparation of control solutions
Respectively and precisely weighing 0.04g of beta-caryophyllene reference substance and 0.01g of matrine reference substance in 10mL volumetric flasks, and adding 50% ethanol to prepare a reference substance solution with the mass concentration of the beta-caryophyllene of 4g/L and the mass concentration of the matrine of 1 g/L.
Sucking 60 mu L of beta-caryophyllene reference substance solution and 300 mu L of matrine reference substance solution which pass through a 0.22 mu m microporous filter membrane into a gas-phase sample injection bottle, respectively adding 100 mu L of internal standard solution 1, 50 mu L of internal standard solution 2 and 50% ethanol for dilution to 1mL, and preparing a mixed reference substance solution.
2.1.5.4 preparation of test solutions
Taking the supernatant prepared by the method of '4.5.1', filtering the supernatant by a 0.22 mu m microporous membrane, sucking 500 mu L of the supernatant into a gas phase sampling bottle, and respectively adding 100 mu L of internal standard solution 1, 50 mu L of internal standard solution 2 and 50% ethanol for diluting to 1ml to prepare a sample solution.
The mass spectrograms of the internal standard substance dodecane, dicyclohexyl phthalate and the standard substance beta-caryophyllene and matrine are shown in figure 2, and the standard substance (a) and the beta-caryophyllene-matrine @ NH are mixed2The flow chart of the total gasmass ion flow of the HMSN microspheres (b) is shown in FIG. 3.
2.1.5.5 methodology examination
2.1.5.5.1 examination of Linear relationships
Respectively sucking 8, 20, 60, 80, 160 and 200 mu L of beta-caryophyllene reference substance solution and 10, 50, 150, 200, 300 and 400 mu L of matrine reference substance solution, placing the solutions into a gas-phase sampling bottle, respectively adding 100 mu L of internal standard solution 1 and 50 mu L of internal standard solution 2, diluting the solutions to 1mL by 50% ethanol, and carrying out sample injection determination according to the chromatographic condition under 2.1.5.1. Taking the ratio of peak areas of beta-caryophyllene and dodecane as a vertical coordinate Y, taking the ratio of content of beta-caryophyllene and dodecane as a horizontal coordinate X, obtaining a linear regression equation Y of 2.091X +0.1058(r of 0.9995), and ensuring that the linear relation of the concentration of beta-caryophyllene is good at 0.032-0.800 mg/mL; taking the peak area ratio of matrine to dicyclohexyl phthalate as the ordinate Y and the content ratio of matrine to dicyclohexyl phthalate as the abscissa X to obtain a linear regression equation Y of 0.6175X +0.1069(r of 0.9995), wherein the matrine concentration is in a good linear relation of 0.010-0.400 mg/mL.
2.1.5.5.2 precision test
Taking a mixed reference substance solution with the concentration of the beta-caryophyllene of 0.72mg/mL and the concentration of the matrine of 0.04mg/mL, continuously injecting samples for 6 times under the chromatographic condition of 2.1.5.1, respectively calculating the peak area ratio of the beta-caryophyllene to the internal standard substance 1 and the peak area ratio of the matrine to the internal standard substance 2 according to the chromatographic result, and obtaining RSD values of 0.84% and 0.68%, which indicates that the precision of the instrument is good.
2.1.5.5.3 stability test
Taking the same batch of sample solution, continuously injecting sample for 6 times under the chromatographic condition of 2.1.5.1 for 0, 2, 4, 6, 8, 12 and 24 hours respectively, calculating the peak area ratio of beta-caryophyllene to internal standard substance 1 and the peak area ratio of matrine to internal standard substance 2 according to the chromatographic result respectively to obtain RSD values of 1.29% and 1.50%, and explaining that the beta-caryophyllene and the matrine are coated with NH2HMSN has good stability within 24 h.
2.1.5.5.4 repeatability test
Taking the same batch of sample solution, carrying out continuous sample injection for 6 times under the chromatographic condition of 2.1.5.1, respectively calculating the peak area ratio of the beta-caryophyllene to the internal standard substance 1 and the peak area ratio of the matrine to the internal standard substance 2 according to the chromatographic result, and obtaining RSD values of 0.58% and 1.53%, which indicates that the method has good repeatability.
2.1.5.5.5 sample recovery test
Accurately sucking 6 parts of sample solution with known content of 500 mu L, adding beta-caryophyllene and matrine with the same content, respectively adding 100 mu L of internal standard solution 1 and 50 mu L of internal standard solution 2, diluting to 1mL by 50% ethanol, performing sample injection measurement under the chromatographic condition of '2.1.5.1', and obtaining the average recovery rates of the beta-caryophyllene and the matrine respectively of 99.73% and 103.87% and RSD of 0.86% and 1.12% according to the chromatographic result (see Table 15).
TABLE 15 test results of sample recovery of beta-caryophyllene and matrine
2.1.5.6 content determination of sample
And (3) enabling the sample solution sampled at regular time to pass through a 0.22-micron microporous filter membrane, accurately sucking 500 mu L of the solution into a gas phase sampling bottle, adding 100 mu L of internal standard solution 1 and 50 mu L of internal standard solution 2, diluting to 1mL by using 50% ethanol, determining according to the chromatographic condition under 2.1.5.1, and calculating the release content of the sample at different sampling time.
2.1.6 measurement of stability of drug-loaded microspheres at different storage temperatures
Reference pesticide heat storage and low temperature stability determination method (GB-T1)9136-2003, GB-T19137-2003), weighing 1g beta-caryophyllene matrine @ NH2Putting HMSN drug-loaded microspheres into a clean brown vial, sealing, putting into a 0 ℃ refrigerator, a room temperature dryer and a 54 ℃ constant temperature box respectively for stability experiments, taking out after storage for 1h to see that no precipitation phenomenon exists, taking out after storage for 14d, preparing into 0.1% solution to be detected, and determining the particle size of the solution.
2.2 drug-loaded microsphere test of drug efficacy
2.2.1 potting test
The test method was the same as the "2.4.1 pot test" described above.
2.2.2 Effect of drug-loaded microspheres on effective ingredients of target leaves
2.2.2.1 sample treatment
Beta-caryophyllene matrine @ NH2And (3) carrying out HMSN (high molecular weight polyethylene glycol-styrene) application treatment, namely taking honeysuckle leaves of 0, 1, 3, 7, 10 and 15 days after application, cleaning, blowing and drying at 50-60 ℃, crushing, sieving with a 60-mesh sieve, sealing, drying and storing.
2.2.2.2 chromatographic conditions
A chromatographic column: agilent ZORBAX XDB-C18 column (250 mm. times.4.6 mm, 5 μm); mobile phase: 0.2% formic acid (a) -acetonitrile (B), elution gradient see table 16, column temperature: 30 ℃, sample introduction: 10 mu L of the solution; detection wavelength: 327nm, 350 nm.
TABLE 16 elution procedure
2.2.2.3 preparation of control solutions
Accurately weighing appropriate amount of reference substance, adding 50% methanol to obtain mixed reference substance solutions with mass concentrations of chlorogenic acid 259.2 μ g/mL, isochlorogenic acid A360.0 μ g/mL, isochlorogenic acid C192.0 μ g/mL, and luteolin 100.0 μ g/mL.
2.2.2.4 preparation of test solution
Precisely weighing 0.2500g of sample, placing the sample into a 25mL conical flask with a plug, precisely adding 25mL of 50% methanol, weighing, performing ultrasonic treatment (100W, 100kHZ), treating for 30min, cooling, weighing again, complementing the weight with 50% methanol solution, shaking uniformly, centrifuging to obtain supernatant, filtering through a 0.22 mu m organic microporous filter head, and taking the subsequent filtrate as a test solution.
The HPLC chromatogram of the solution obtained by mixing the control (A) and the test (B) is shown in FIG. 4.
2.2.2.5 content determination of sample
Taking the sample powder of different treatments, preparing sample solution according to the method under '2.2.2.4', carrying out sample injection determination under the condition of '2.2.2.2', analyzing chromatogram, and calculating to obtain the content change of chlorogenic acid, luteoloside, isochlorogenic acid A and isochlorogenic acid C in the honeysuckle leaves.
2.2.2.6 methodology examination
2.2.2.6.1 inspection of linear relationship
Taking a proper amount of mixed reference substance solution, preparing into mixed reference substance solution with series concentration with 50% methanol solution, performing sample injection determination under the chromatographic condition of 2.2.2.2, and analyzing chromatogram. The results of regression analysis were shown in Table 17, using the concentration (. mu.g/mL) of the control solution as the abscissa and the peak area as the ordinate.
TABLE 17 Linear relationship of 4 ingredients of Lonicera japonica leaf
2.2.2.6.2 precision test
Taking a proper amount of the mixed reference solution, continuously injecting sample for 6 times according to the chromatographic condition under 2.2.2.2, and analyzing the chromatogram. The RSD of the peak areas of the chlorogenic acid, the luteoloside, the isochlorogenic acid A and the isochlorogenic acid C obtained after calculation are respectively 0.33%, 0.14%, 0.55% and 0.53%, which indicates that the precision of the instrument is good.
2.2.2.6.3 repeatability test
Taking 6 parts of the same test sample, each 0.25g, preparing a test sample solution according to the method under the condition of 2.2.2.4, injecting sample according to the chromatographic condition under the condition of 2.2.2.2, and analyzing the chromatogram. The RSD of the peak areas of chlorogenic acid, galuteolin, isochlorogenic acid A and isochlorogenic acid C obtained by calculation are respectively 1.10%, 1.44%, 1.29% and 1.21%, which indicates that the method has good repeatability.
2.2.2.6.4 stability test
Sampling the same batch of sample solution under chromatographic conditions of 2.2.2.2 for 0, 2, 4, 6, 8, 12 and 24h respectively, and analyzing chromatogram. The RSD of the peak areas of chlorogenic acid, galuteolin, isochlorogenic acid A and isochlorogenic acid C are respectively 0.14%, 0.19%, 0.30% and 0.39% by calculation, which indicates that the test solution is stable within 24 h.
2.2.5.6.5 sample recovery test
Respectively taking 6 parts of test sample with known component content, accurately weighing 0.25g of each part, adding chlorogenic acid, luteolin, isochlorogenic acid A and isochlorogenic acid C with the same amount, preparing test sample solution according to the method under 2.2.2.4, injecting sample according to the chromatographic condition under 2.2.2.2, and analyzing chromatogram. The average recovery rates of chlorogenic acid, galuteolin, isochlorogenic acid A and isochlorogenic acid C were calculated to be 101.85%, 100.82%, 100.11% and 101.27%, respectively, and the RSD corresponding to the peak areas were 1.18%, 1.49%, 1.28% and 1.15%, respectively (see Table 18).
Table 184 sample application recovery test results (n ═ 3)
2.3 drug-loaded microsphere potentiation study
In order to ensure the consistency of materials and reduce experimental errors, the honeysuckle selected in the experiment uses the same culture conditions in a greenhouse, and the growth periods are consistent, so the difference caused by different materials can be ignored in the experiment. Collecting honeysuckle leaves in a vigorous growth period, washing floating dust on the leaves with deionized water, paying attention to not destroy the structural form of the honeysuckle leaves, and airing the honeysuckle leaves in a natural state for later use (see figure 5).
2.3.1 wettability determination
According to the literature, a part between a main vein and an edge of honeysuckle is cut by a scalpel to be used as a sample, the sample is fixed on a glass slide by a double-sided adhesive tape, then the sample is placed on an instrument objective table, 5 mu L of deionized water or a sample to be measured is absorbed and uniformly dripped on the honeysuckle leaf, when the liquid dripping state is stable, an image is captured and a numerical value is recorded, and an average value is obtained by measuring for 3 times to ensure the accuracy.
2.3.2 deposition Performance determination
Wrapping the treated honeysuckle leaves with preservative film along the main veins, dividing the leaves into two parts, and using beta-caryophyllene, matrine and NH2Uniformly spraying the HMSN solution, collecting leaves after the leaves are naturally dried, taking off a preservative film, cutting the preservative film into two halves along the main leaf vein by using a scalpel, placing one half on clean filter paper, washing the other half by using deionized water, placing the washed half in an oven for 6 hours, taking out the gold spraying, and observing the deposition phenomenon of the drug-loaded microspheres on the honeysuckle leaves under a scanning electron microscope.
3 results and analysis
3.1 beta-caryophyllene matrine @ NH2Preparation and characterization of HMSN
3.1.1 drug-loaded microsphere preparation conditions and morphology observation
From the SEM image, it can be clearly observed that the synthesized silica microspheres (a) are uniform in size and are in a solid spherical shape; the hollow silica microspheres (b) are in a hollow structure after being broken, the wall of each microsphere has a certain thickness, and the overall morphology of the microspheres is not much different from that of the silica microspheres (see fig. 6). A vermicular pore passage structure is shown after MSN is calcined and CTAB is removed under a transmission electron microscope, the hollow structure and the spherical wall thickness of HMSN are obviously visible, and the vermicular pore passage structure is more orderly (see figure 7).
3.1.1.1 observation of morphology of silica microspheres with CTAB
CTAB dosage mainly influences the thickness of the sphere wall and the dispersion degree of the formed microspheres, TEOS dosage in the experiment is 2mL, CTAB dosage is 0.15mL, 0.3mL, 0.4mL and 0.6mL respectively, and the morphology of the synthesized silica microspheres is observed under SEM. When the CTAB dosage is 0.15mL, the synthesized silicon dioxide microspheres are uniform in size and good in dispersity; when the CTAB dosage is 0.3mL, the microspheres are uniform in size and slightly adhered to each other; when the CTAB dosage is 0.4mL or 0.6mL, the microspheres are seriously adhered and have uneven sizes, because the positive micelles formed by the large CTAB dosage are more, and the hydrolyzed negative charges are quickly combined with the positive micelles under the electrostatic adsorption action after the TEOS is added, so that the microspheres are multiply adhered and have poor dispersibility. Therefore, the CTAB amount ratio in the silica microsphere synthesis reaction was 0.3mL (see FIG. 8).
3.1.1.2 influence of ammonia water dosage on shape of silicon dioxide microsphere
The ammonia water dosage can influence the concentration of OH < - > in a reaction system, OH < - > can directly initiate nucleophilic attack, the TEOS hydrolysis speed is influenced, more micronuclei are generated, the microsphere polycondensation reaction is promoted, and the particle size of the generated microsphere is increased. The ammonia water dosage in the experiment is 1, 2, 3 and 4mL respectively, and the morphology of the synthesized microsphere is shown in FIG. 9. When the ammonia water consumption is 1 and 2mL, the particle size difference of the microspheres is not large, but the dispersibility is better when the ammonia water consumption is 2 mL; the ammonia water dosage is 3mL or 4mL, the OH-concentration is increased, the TEOS hydrolysis speed is high, the formed silicon dioxide microspheres are uneven, the particle size difference is large, and the agglomeration phenomenon is obvious. Therefore, the amount of ammonia used in the reaction system was 2 mL.
3.1.1.3 Effect of temperature on morphology of silica microspheres
The temperature can directly influence the nucleation rate of the microspheres or influence the volatilization speed of ammonia water in the system, thereby influencing the TEOS hydrolysis speed and the nucleation rate of the silicon dioxide microspheres. When the temperature of the reaction system is room temperature and 35 ℃, the particle size of the silicon dioxide microspheres is not greatly different, but the agglomeration phenomenon of the microsphere particles is obvious along with the temperature rise; when the temperature of the reaction system is 80 ℃, the particle size of the silicon dioxide microspheres is reduced, but most microspheres are agglomerated together, and the dispersibility is extremely poor. The synthesis of silica microspheres was therefore all carried out at room temperature (see figure 10).
In conclusion, the optimal preparation conditions for obtaining the hollow silica microspheres are as follows: 0.3000g (accurate to 0.0002) CTAB is dissolved in the mixed solution (60mL of absolute ethyl alcohol and 100mL of deionized water), the mixture is magnetically stirred, 2mL of strong ammonia water is added, 2mL of TEOS is added dropwise after the mixture is uniformly mixed, and the reaction is carried out for 6 hours at room temperature. Washing with deionized water and anhydrous ethanol for 3 times, centrifuging, dispersing the white precipitate into a mixed solution of 60mL of deionized water, 60mL of anhydrous ethanol and 9mL of cyclohexane, sealing, placing in an oven for 3d, centrifuging, washing, and vacuum drying. Calcining at 550 ℃ for 6h in a muffle furnace, removing CTAB, and standing at room temperature under drying to obtain HMSN.
3.1.2 Fourier Infrared Spectroscopy
As can be seen from FIG. 11, β -caryophyllene matrine @ NH2HMSN at 456cm-1、805cm-1The peak is the symmetric stretching vibration peak of Si-O bond, 1070cm-1The strong and wide peak is the antisymmetric stretching vibration of Si-O-Si; 3275cm-1The absorption peak is caused by the mutual overlapping of the asymmetric stretching vibration peak of N-H and the Si-OH peak in the silicon dioxide, which indicates that the amino group is modified on the HMSN and is positioned at NH2-no characteristic peak of CTAB was found in the ir spectrum of HMSN, indicating that the surfactant had been removed. 1617cm-1Is the stretching vibration peak of C ═ O, 1455cm-1Has a skeleton of C (2928 cm)-1And 2800cm-1Left and right have-CH2Asymmetric and symmetric stretching vibration peaks of (2) illustrate NH2the-HMSN has been successfully loaded with beta-caryophyllene matrine.
3.1.3 particle size and potential analysis
According to the determination result, the MSN has an average particle size of about 540nm and a Zeta potential of about-19.52; the HMSN modified by the amino has the average particle size of about 554nm, the Zeta potential of about 27.67 and slightly increased particle size, which is probably caused by the reaming of cyclohexane and the amino modification process, and the positive potential is caused by the modification of positively charged amino particles on the surface of the microsphere; the average particle size of the drug-loaded microspheres is about 628nm, the Zeta potential is about 47.79, the increase in particle size is caused by agglomeration of the drug-loaded microspheres, and the increase in potential is caused by drug loading (see table 19).
TABLE 19 MSN, NH2-HMSN and beta-caryophyllene matrine @ NH2Average particle size and zeta potential of HMSN
3.1.4X-ray diffraction (XRD) analysis
NH2-HMSN and beta-caryophyllene matrine @ NH2The XRD pattern of HMSN drug-loaded microspheres is as follows (see fig. 12). NH (NH)2HMSN has a dome peak around 24 ° and no distinct crystalline diffraction peak, which is a characteristic peak of amorphous silica; beta-caryophyllene matrine @ NH2-HMSN and NH2The X-ray diffraction patterns of the HMSN are basically consistent, only one dome peak is provided, and the result shows that the beta-caryophyllene matrine is completely loaded in the microspheres in an amorphous state, and the structure of the microspheres is not damaged in the loading process.
3.1.5 drug load determination
As can be seen from FIG. 13, as the drug loading ratio increased, β -caryophyllene matrine @ NH2The drug loading of HMSN increases gradually. When the drug loading ratio is 1:1 and 2: beta-caryophyllene-matrine @ NH prepared at 1 time2The drug loading rate of the HMSN is not very different, and the drug loading rates are 23.38 percent and 24.05 percent of beta-caryophyllene and 10.96 percent and 11.20 percent of matrine respectively. In combination with cost considerations, the ratio of drug to carrier was chosen experimentally as 1:1, preparing drug-loaded microspheres.
3.1.6 Thermogravimetric (TG) analysis
By NH2Thermogravimetric curves of-HMSN (see FIG. 14), NH2The thermal stability of-HMSN is better and NH is not higher than 100 DEG C2The mass loss of HMSN is evident from the loss of adsorbed water in the sample, after which the temperature rise mass loss is hardly changed. The mass loss of the physical mixture of the medicament and the microspheres is obvious below 230 ℃, which is caused by the adsorbed water in the sample and the decomposition of beta-caryophyllene-matrine on the surfaces of the microspheres, and then the mass is hardly lost along with the temperature rise, which indicates that the medicament is not loaded in the microspheres at this time. Beta-caryophyllene matrine @ NH2The mass loss of HMSN is mainly caused by the loss of adsorbed water in the sample before 100 ℃; the mass loss at 100-250 ℃ is the pyrolysis of beta-caryophyllene and matrine adsorbed on the surface of a sample; the mass loss at 250-420 ℃ is that beta-caryophyllene and matrine loaded in the drug-carrying microspheres are decomposed; the mass loss is almost unchanged at the temperature of above 420 ℃, and the 4 weight loss processes further explain that the beta-caryophyllene matrine is loaded in the microspheres. Calculated to obtain NH above 100 DEG C2-HMSN, beta-caryophyllene matrine @ NH2The mass loss rates of the physical mixture of HMSN, microspheres and pharmaceutical agent were 4.45%, 40.58%, 42.25%, respectively. Meanwhile, the drug-loading rate of the drug-loaded microspheres can be calculated to be 36.13% according to the mass loss rate.
3.2 measurement of sustained Release Performance
The hollow silica microspheres are of a structure with a shell layer wrapping and a hollow interior, most of the medicine is unevenly distributed after entering the hollow silica microspheres to form concentration difference, the concentration difference inside and outside the hollow silica microspheres at the initial stage of release is large, the medicine release rate is high, the medicine-carrying microspheres tend to be slow in release rate along with the increase of time, and the sustained-release effect is achieved. As can be seen from fig. 15, the release rate of the compound raw pesticide is relatively high, the release rate is linearly increased, and the cumulative release rates of the beta-caryophyllene and the matrine in 48 hours are respectively 80.02% and 76.77%; and beta-caryophyllene matrine @ NH2the-HMSN can be slowly released in ethanol-water, the cumulative release rates of the beta-caryophyllene and the matrine in 288h are respectively 88.85 percent and 84.24 percent, the slow release effect is obvious, and the drug effect time can be prolonged. The sustained-release time is used as an abscissa, the accumulated drug release rate is used as an ordinate, and the beta-caryophyllene release kinetic model obtained through origin9.0 fitting analysis is y-86.631 e-0.00129x +91.799(r is 0.992), the matrine release kinetic model is y-91.105 e-0.00797x +04.293(r is 0.994), the correlation coefficients are all larger than 0.99, and the first-order kinetic model is met.
3.3 beta-caryophyllene matrine @ NH2Stability analysis of HMSN
The stability has important significance on the drug effect, and is one of evaluation indexes of pesticide formulations, based on the evaluation indexes, the experiment determines the particle size and the morphology of the drug-loaded microspheres at different storage temperatures, and the determination results are as follows: after the drug-loaded microspheres are stored for 14 days at 0 ℃, room temperature and 54 ℃, the average particle diameters are 631.29 +/-12.01, 629.83 +/-13.24 and 621.94 +/-19.86 respectively, the average particle diameters of the drug-loaded microspheres at 0 ℃ and room temperature do not change greatly, which shows that the stability of the drug-loaded microspheres at the temperature is good, the average particle diameters of the drug-loaded microspheres slightly change after the drug-loaded microspheres are stored for two weeks at 54 ℃, and the change of the particles is probably caused by the loss of residual drugs on the surfaces of the drug-loaded microspheres due to the increase of the temperature. Therefore, the drug-loaded microspheres have good stability at 0 ℃ and room temperature and can be stored for a long time.
3.4 potting test
As can be seen from Table 20, as compared with the beta-caryophyllene matrine complex formulation, beta-caryophyllene matrine @ NH2Quick-acting HMSN for controlling honeysuckle aphidThe pesticide has the advantages of quick response, high control effect and long lasting period, the control effect is approximately consistent with that of 10 percent imidacloprid EC of chemical pesticide after the pesticide is applied for 3 days, and the control effect can reach 81 percent by 500 times of liquid correction; the control effect shows an increasing trend after 1-15 days of application, the correction control effect is about 94.87% and 96.55% after 10 days and 15 days of application, and the lasting period is better than 10% imidacloprid EC. The pharmacodynamic test shows that the medicine is prepared by NH2The beta-caryophyllene and matrine loaded with the HMSN has quick response, high prevention and treatment effect on honeysuckle aphids, slow release effect and high efficiency and lasting effect.
Potted plant experiment of table 20 drug-loaded microspheres for preventing and treating honeysuckle aphids
Note: letters following the same column of numbers indicate significant differences at the P < 0.05 level by Duncan's New Complex Pole Difference method.
3.5 Effect of drug-loaded microspheres on effective ingredients of target leaves
As can be seen from FIG. 16, the contents of the 4 effective components of the target leaves are all reduced after the aphids damage the target leaves, and the differences are obvious (P is less than 0.05). The contents of chlorogenic acid and luteolin after 1d application are increased compared with the contents after insect attack, but are still lower than the normal leaf contents; after the pesticide is applied for 3d, the content of isochlorogenic acid A is obviously increased compared with that of the pesticide, and the content of chlorogenic acid is restored to a normal level; the content of chlorogenic acid and isochlorogenic acid C is slightly higher than that of normal leaves after the medicine is applied for 10 days, and the content of luteolin and isochlorogenic acid A is similar to that of the normal leaves; the content of chlorogenic acid is slightly reduced after 15 days of administration, but is similar to that of normal leaves, and the content of the other 3 components is higher than that of the normal leaves. In a word, the content of the 4 effective components of the target leaves is obviously reduced after the aphids damage the target leaves, and the content of the 4 effective components after the pesticide application treatment is increased along with the prolonging of the pesticide application time and gradually returns to the normal level, which also indicates that the pesticide has certain control effect on the aphids and can repair the damage to the honeysuckle leaves caused by the damage of the aphids.
3.6 wetting Performance analysis
By enhancing the adhesion, wettability and wetting of the pesticide formulation on the surface of the leafThe deposition amount is an important way for improving the utilization rate and the pesticide effect of the pesticide. The wettability of the leaf surface shows the affinity of the leaf blade to water, and can be determined by measuring the contact angle of an air interface, a solid interface and a liquid interface, the contact angle theta is smaller than 110 degrees for wetting, the theta is larger than 130 degrees for non-wetting, the water repellency is shown, the liquid medicine can spread to form a film on the leaf surface with strong wettability, and the medicine beads can roll off on the non-wetting leaf surface. The experiment determines beta-caryophyllene raw drug, matrine raw drug, beta-caryophyllene and matrine compound agent, beta-caryophyllene and matrine @ NH2The contact angle and the infiltration behavior of HMSN and water on the honeysuckle leaf surfaces are that the contact angle of the beta-caryophyllene raw drug is 91.27 +/-1.46 which is measured as oily liquid and is smaller than the contact angle of the water and matrine raw drug on the honeysuckle leaf surfaces; the contact angle of the beta-caryophyllene and matrine compound agent is 87.33 +/-1.99, which is reduced compared with the contact angle of the original drug, and the beta-caryophyllene and matrine @ NH2The HMSN contact angle value is 81.97 +/-1.85, the contact angle is the smallest compared with that of other medicaments on the leaf surfaces of honeysuckle, the difference is obvious (P is less than 0.05), and the wettability of the compound preparation is greatly improved after the compound preparation is loaded by microspheres (see figure 17 and table 21).
TABLE 21 average contact angle of different agents on honeysuckle leaves
3.7 deposition Performance analysis
The effective utilization rate of the pesticide is closely related to the deposition performance of the pesticide liquid on the surface of the leaf besides the adhesiveness and wettability of the pesticide liquid on the target leaf surface. In order to truly simulate the deposition of the liquid medicine on the honeysuckle leaves, the honeysuckle leaves are shot by an environmental scanning electron microscope, and as can be seen from figure 18, a large amount of beta-caryophyllene-matrine @ NH still remains after the honeysuckle leaves are washed by water for multiple times2The HMSN drug-carrying microspheres are retained on villi, waxy layers and intercellular spaces on the surfaces of the leaves, which indicates that the beta-caryophyllene-matrine@NH2The HMSN drug-carrying microspheres have the capability of affinity deposition on the honeysuckle leaves.
4. Discussion and summary
The invention takes 17 percent beta-caryophyllene and matrine with obvious control effect as an object, uses hollow silicon dioxide microspheres to load a compound agent to prepare the slow-release drug-carrying microspheres, selects honeysuckle leaves as a target model, and researches the representation, the slow-release performance, the synergistic mechanism and the like of the drug-carrying microspheres. The results are as follows:
the beta-caryophyllene-matrine @ NH is prepared by experiments2The optimal conditions of the HMSN drug-loaded microspheres are as follows: 0.3000g CTAB is dissolved in the mixed solution (60mL of absolute ethyl alcohol and 100mL of deionized water), the mixture is magnetically stirred, 2mL of strong ammonia water is added, 2mL of TEOS is dropwise added after the mixture is fully mixed, and the reaction is carried out for 6 hours at room temperature. And (4) reaming the washed and centrifuged white precipitate by cyclohexane, and calcining by a muffle furnace to obtain the HMSN. After amino modification, according to the proportion of beta-caryophyllene-matrine: NH (NH)2-HMSN mass ratio of 1:1 preparing medicine carrying microsphere, and the medicine carrying amount is about 36.13% under the condition. The drug-loaded microspheres have obvious slow release effect, the cumulative release rate after 288h is more than 80 percent, the slow release of the drug can be realized, and the lasting period of the drug is prolonged.
The pharmacodynamic test shows that the beta-caryophyllene-matrine @ NH2The HMSN has good quick-acting performance and high drug effect in preventing and treating honeysuckle aphids, can continuously exert the drug effect, and can effectively inhibit the honeysuckle aphid population growth. The correction control effect of the drug-loaded microspheres after being applied for 10 days is more than 90 percent, the drug-loaded microspheres have no obvious difference compared with 10 percent imidacloprid missible oil, the correction control effect can reach 96 percent after being applied for 15 days under 500 times of liquid, and the correction control effect is obviously higher than that of beta-caryophyllene-matrine and 10 percent imidacloprid missible oil. According to the slow release characteristics of the drug-loaded microspheres, the drug-loaded microspheres are recommended to be used when the population base numbers of honeysuckle aphids are small, such as the non-occurrence time and the initial occurrence time of the honeysuckle aphids, and can be used for controlling the aphids for a long time. After the target leaves are collected and applied with the pesticide, the content of 4 effective components in the leaves is reduced after the aphid is damaged, and the content of the four components gradually recovers to the normal leaf level along with the increase of the pesticide application time after the pesticide carrying microspheres are sprayed. This also describes β -caryophyllene matrine @ NH2The HMSN can slowly release the pesticide effect, effectively prevent and treat honeysuckle aphid and can repair aphid pairsDamage to the target leaf.
The wax layer of the leaf contains a large amount of lipophilic compounds and carboxyl, aldehyde and glycosidic bond groups, so that the leaf has hydrophobicity and negative charge, and beta-caryophyllene-matrine @ NH2-NH modified on surface of HMSN drug-carrying microsphere2The solution has positive charge, can be in hydrogen bond with-OH in the wax layer, can be in electrostatic adsorption with-COOH and can be in covalent bonding with-CHO. A series of complex interaction mechanisms can enhance the affinity of the drug-loaded microspheres on the target leaf surface and improve the wettability and retention capacity of the drug-loaded microspheres on the target leaf surface.
Fifth, summarize
The aphids are one of the most serious pests in the honeysuckle planting process, the breeding speed is high, the aphids feed the honeysuckle juice to grow, so that leaves curl, turn yellow and even fall off, buds are damaged to cause growth retardation, and the yield and the quality of the honeysuckle are seriously affected. With the increasing prominence of the use defects of chemical pesticides, the research and development of efficient, safe and green plant-source pesticides gradually become a hotspot of research. The invention determines the influence of 21 botanical substances on the biological activity of honeysuckle aphids, carries out compound research on the screened botanical substances, prepares the slow-release drug-carrying microspheres, and discusses a synergistic mechanism so as to obtain safe, efficient and lasting botanical pesticides and effectively control the honeysuckle aphids. The main study results are as follows:
1. systematic investigation is carried out on the occurrence rules of honeysuckle aphids in four states, namely eggs, nymphs, adult aphids and winged aphids, and basic guidance is provided for staged pesticide application and effective prevention and control.
2. The contact poisoning and stomach poisoning activities of 21 plant source substances on honeysuckle aphids are determined, and 5 plant source substances with higher contact poisoning and stomach poisoning activities are screened out: thymol, beta-caryophyllene, trans-cinnamaldehyde, matrine and azadirachtin. 5 the toxicity of the plant source materials is matrine, nimbin, beta-caryophyllene, trans-cinnamaldehyde and thymol respectively.
3.3 kinds of compound combinations with obvious synergistic effect are screened out by utilizing a co-toxicity factor and co-toxicity coefficient method and combining with an SPSS mathematical fitting model, and the compound agent of 16 percent of thymol, beta-caryophyllene, 17 percent of beta-caryophyllene, matrine and 13 percent of trans-cinnamic aldehyde and azadirachtin is prepared respectively. Compared with the original drugs, the 3 compound agents have enhanced toxicity to honeysuckle aphids, and 17 percent of beta-caryophyllene, matrine and 13 percent of trans-cinnamic aldehyde, azadirachtin have obvious synergistic effect on the respective original drugs. The honeysuckle aphid prevention and control effect is better and the prevention and control effect is higher by using 17 percent of beta-caryophyllene and matrine in the 3 compound agents.
4. The sustained-release drug-carrying microspheres are prepared by loading the compound preparation with the hollow silica microspheres for the first time, so that the active ingredients are effectively protected, the 288h cumulative release rate is more than 80%, the sustained-release performance is good, and the defects of the traditional pesticide dosage form are overcome. Beta-caryophyllene matrine @ NH2The HMSN is quick in quick-acting property, high in control effect, obvious in slow release effect and long in lasting period in controlling honeysuckle aphids, the control effect is still as high as 96% after 15d of pesticide application, and honeysuckle aphids can be controlled efficiently and durably. In addition, beta-caryophyllene matrine @ NH2The HMSN has a repairing effect on damaged honeysuckle leaves, and the content of active ingredients of target leaves can be gradually restored to the level of normal leaves within 10-15 days after the application.
5. Beta-caryophyllene matrine @ NH2The HMSN can improve the affinity of the liquid medicine and the target leaf surface, the liquid medicine loss is small during the application, the utilization rate is high, the medicine effect can be effectively exerted, and the synergistic effect is obvious.
The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. The compound synergistic composition is characterized in that the compound synergistic composition is any one of trans-cinnamic aldehyde and azadirachtin; wherein the mass ratio of the trans-cinnamaldehyde to the azadirachtin is 0.45:1-4.2: 1.
2. The compound synergistic composition as claimed in claim 1, wherein the mass ratio of trans-cinnamaldehyde to azadirachtin is 9:20, 27:35, 6:5, 9:5, 27:10, 21:5 or 1.6: 1.
3. The compound synergistic composition as claimed in claim 2, wherein the trans-cinnamaldehyde azadirachtin is 13% by mass of the trans-cinnamaldehyde azadirachtin compound, the trans-cinnamaldehyde is 8% by mass, and the azadirachtin is 5% by mass.
4. An insecticide compound drug-carrying microsphere applying the compound synergistic composition as claimed in claims 1 to 3, wherein the effective component of the insecticide compound drug-carrying microsphere is the compound synergistic composition as claimed in any one of claims 1 to 3.
5. The preparation method of the pesticide compound drug-loaded microsphere according to claim 4, which is characterized by comprising the following steps:
step one, preparing hollow silica microspheres HMSN;
step two, amino-modified hollow silica microsphere NH is carried out2-preparation of HMSN;
step three, carrying out beta-caryophyllene-matrine @ NH2Preparation of HMSN drug-loaded microspheres.
6. The preparation method of the pesticide compound drug-loaded microsphere according to claim 5, wherein in the first step, the preparation of the hollow silica microsphere HMSN comprises the following steps:
(1) weighing a certain amount of hexadecyl trimethyl ammonium bromide, dissolving the hexadecyl trimethyl ammonium bromide in an ethanol solution, magnetically stirring, adding concentrated ammonia water, fully mixing, dropwise adding tetraethyl orthosilicate, and reacting for 6 hours at room temperature;
(2) washing with deionized water and anhydrous ethanol for 3 times, centrifuging, dispersing the white precipitate into mixed solution, sealing, placing in oven for 3d at 60 deg.C, vacuum filtering, washing with deionized water for 3 times, and freeze drying;
(3) placing into a muffle furnace, calcining at 550 deg.C for 6h, removing hexadecyl trimethyl ammonium bromide, and storing at room temperature under drying condition, and recording as HMSN.
7. The preparation method of the pesticide compound drug-loaded microsphere as claimed in claim 6, wherein in the step (1), the amount of tetraethyl orthosilicate is 2mL, the amount of cetyl trimethylammonium bromide is 0.15-0.3mL, and the amount of concentrated ammonia water is 1-2 mL.
8. The preparation method of the pesticide compound drug-loaded microsphere according to claim 5, characterized in that in the third step, the beta-caryophyllene-matrine @ NH2-preparation of HMSN drug-loaded microspheres comprising:
(1) accurately weighing 100mg of 17% beta-caryophyllene and matrine, adding appropriate amount of ethanol, ultrasonic treating to dissolve the medicinal preparation, preparing into medicinal liquid with mass concentration of 5mg/L, and heating in 60 deg.C water bath;
(2) according to beta-caryophyllene, matrine and NH2-adding NH into HMSN with the mass ratio of 1:1-2:12Carrying out ultrasonic treatment for 30min to fully disperse HMSN, stirring for 3h in a sealed condition, continuously stirring for 1h in an open condition, and volatilizing an organic solvent;
(3) washing residual agent with hot ethanol, and freeze drying to obtain beta-caryophyllene-matrine @ NH with different proportions2-HMSN drug loaded microspheres.
9. The preparation method of the pesticide compound drug-loaded microsphere according to claim 8, which is characterized in that in the third step, the ratio of beta-caryophyllene-matrine: NH (NH)2-HMSN mass ratio of 1: 1.
10. the application of the insecticide compound drug-loaded microsphere as claimed in claim 4 in prevention and treatment of honeysuckle aphids.
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| CN114467963B (en) | 2024-02-13 |
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