A kind of double-loading microcapsule containing pyraclostrobin and brassinolide and its preparation method and application

Abstract

The application provides a kind of double-loading microcapsule containing pyraclostrobin and brassinolide and its preparation method and application, and the double-loading microcapsule includes core material and wall material, and the wall material covers the core material;The core material includes pyraclostrobin and 24-epibrassinolide, and the wall material includes polylactic acid-hydroxyacetic acid copolymer and hydroxypropyl methyl cellulose phthalate, and the mass ratio of polylactic acid-hydroxyacetic acid copolymer, hydroxypropyl methyl cellulose phthalate is (40-50):(90-100);The application uses polylactic acid-hydroxyacetic acid copolymer and hydroxypropyl methyl cellulose phthalate with suitable mass ratio as wall material, combined with the preferred preparation method, so that the drug loading of microcapsule and the drug encapsulation efficiency are high, the drug diffusion rate can be effectively controlled, the sustained-release effect is good, and the double-loading microcapsule particle size is suitable, the effective period is long, and the preparation method is simple, so that the prepared double-loading microcapsule particle size distribution is uniform, without damage and coating phenomenon.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pesticides, and particularly relates to a double-loading microcapsule containing pyraclostrobin and brassinolide as well as a preparation method and application thereof. BACKGROUND

[0002] Plant diseases are the main factors affecting the yield and quality of agricultural products. At present, the prevention and treatment of plant diseases is mainly based on the concept of Western medicine, that is, using fungicides to kill harmful pathogens. However, the use amount of fungicides is large, and the control effect is poor. Chinese medicine emphasizes improving the body's resistance to resist pathogen invasion, but the treatment effect of Chinese medicine is slow. Therefore, the combination of traditional Chinese medicine and Western medicine is an effective method for disease control at present.

[0003] Pyraclostrobin is a kind of methoxy acrylate fungicide developed by BASF in Germany in 1993, which has good penetration and local systemic activity, and can effectively prevent the growth and spread of pathogenic bacteria, and has both fungicidal effect and protective and therapeutic effect on plants. Brassinolide is a kind of broad-spectrum and efficient plant growth regulator isolated from rape pollen by Mitchell et al., which is called the sixth plant hormone, and has the effects of accelerating plant metabolism and inducing the expression of plant disease resistance genes.

[0004] Pesticide microcapsule is a kind of microcapsule with semi-permeable capsule membrane, which is prepared by chemical, physical or physical-chemical methods, with natural or artificially synthesized high molecular material as wall material and pesticide active substance as core material. Microcapsule can control the release speed of pesticide, prevent degradation of pesticide, improve the persistence of pesticide, and reduce the acute toxicity of pesticide.

[0005] At present, the main dosage form of pyraclostrobin is emulsifiable concentrate and microcapsule suspension, and brassinolide is soluble concentrate. They are often used together to prevent and control diseases and improve yield. However, there is little report on the double-loading microcapsule of pyraclostrobin and brassinolide which can control the release speed. However, for plants with long growth period such as tea fruits (the growth period is as long as 13 months), atractylodes and polygonatum, multiple use is needed to achieve good control effect. Therefore, the application needs to prepare a double-loading microcapsule containing pyraclostrobin and brassinolide, so as to prolong the persistence of them and achieve better control effect. SUMMARY

[0006] The technical problem solved by the present application is to overcome the deficiencies and defects mentioned in the above background art, and to provide a kind of double-loaded microcapsule containing pyraclostrobin and brassinolide and its preparation method and application, using suitable mass ratio of polylactic acid-glycolic acid copolymer and hydroxypropyl methyl cellulose phthalate as wall material, combined with the preferred preparation method, the drug loading and drug encapsulation efficiency of microcapsule are high, the rate of drug diffusion can be effectively controlled, the sustained-release effect is good, and the particle size of the double-loaded microcapsule is suitable, so that the double-loaded microcapsule has a long effective period, and the preparation method is simple, so that the particle size distribution of the prepared double-loaded microcapsule is uniform, without damage and coating phenomenon.

[0007] To solve the above technical problems, the technical solution provided by the present application is:

[0008] A kind of double-loaded microcapsule containing pyraclostrobin and brassinolide, the double-loaded microcapsule includes core material and wall material, the wall material covers the core material;The core material includes pyraclostrobin and 24-epibrassinolide, and the wall material includes polylactic acid-glycolic acid copolymer and hydroxypropyl methyl cellulose phthalate.

[0009] Preferably, the mass ratio of polylactic acid-glycolic acid copolymer and hydroxypropyl methyl cellulose phthalate is (40-50):(90-100). Further preferably, the mass ratio of polylactic acid-glycolic acid copolymer and hydroxypropyl methyl cellulose phthalate is (45-46):(94-95). More preferably, the mass ratio of polylactic acid-glycolic acid copolymer and hydroxypropyl methyl cellulose phthalate is 46:94.

[0010] The present application uses polylactic acid-glycolic acid copolymer and hydroxypropyl methyl cellulose phthalate with suitable mass ratio to prepare the wall material based on pyraclostrobin and 24-epibrassinolide as the core material, both of which have good biocompatibility, excellent capsule and film forming properties, which can effectively control the drug diffusion rate and have good sustained-release effect, and the particle size of the double-loaded microcapsule prepared is suitable and the particle size distribution is uniform, further prolonging the effective period of the double-loaded microcapsule.

[0011] Preferably, the number average molecular weight of the polylactic acid-glycolic acid copolymer is 9000-13000. The use of polylactic acid-glycolic acid copolymer with this number average molecular weight further enhances the capsule and film forming properties of the wall material. Further preferably, the number average molecular weight of the polylactic acid-glycolic acid copolymer is 10000.

[0012] Preferably, the particle size of the double-loaded microcapsule is in the range of 0.75-5.5 μm.

[0013] As a general inventive concept, the present application provides a method for preparing a double-loaded microcapsule containing pyraclostrobin and brassinolide, comprising the following steps:

[0014] (1) dissolving 24-epibrassinolide in a mixture of acetone and water to obtain an inner aqueous phase;

[0015] (2) dissolving polylactic acid-glycolic acid copolymer in dichloromethane to obtain a polylactic acid-glycolic acid copolymer solution; adding hydroxypropyl methylcellulose phthalate into acetone, ultrasonic treatment, then adding the polylactic acid-glycolic acid copolymer solution and continuing ultrasonic treatment to obtain a wall material solution; adding pyraclostrobin into the wall material solution and ultrasonic treatment to obtain an oil phase;

[0016] (3) slowly adding the inner aqueous phase into the oil phase, ultrasonic dispersing for 4-5 s each time, standing for 9-12 s, and ultrasonic dispersing for a total of 3-4 min to obtain a W1 / O emulsion;

[0017] (4) adding the W1 / O emulsion into a polyvinyl alcohol aqueous solution (an outer aqueous phase), ultrasonic dispersing for 3-4 s each time, standing for 3-4 s, and ultrasonic dispersing for a total of 5-7 min to obtain a W1 / O / W2 emulsion;

[0018] (5) performing rotary evaporation on the W1 / O / W2 emulsion to remove acetone and dichloromethane, obtaining a microcapsule emulsion; centrifugally washing the microcapsule emulsion, and then performing vacuum freeze-drying to obtain a double-loaded microcapsule containing pyraclostrobin and brassinolide.

[0019] In steps (3) and (4), the above ultrasonic dispersing process is performed, which can make the formed W1 / O / W2 emulsion system more stable, fully wrap the core material with the wall material, improve the drug loading and encapsulation efficiency, and improve the uniformity of the microcapsule particle size distribution, so that the adhesion phenomenon between the microcapsules is significantly reduced.

[0020] Preferably, in the inner aqueous phase of step (1), the mass-volume ratio of 24-epibrassinolide, acetone and water is (7-70) mg:(4-5) mL:(2-3) mL; and in the oil phase of step (2), the mass-volume ratio of pyraclostrobin, polylactic acid-glycolic acid copolymer, hydroxypropyl methylcellulose phthalate, dichloromethane and acetone is (60-240) mg:(40-50) mg:(90-100) mg:4 mL:1 mL.

[0021] Preferably, in step (4), the concentration of the polyvinyl alcohol aqueous solution is 1% w / v, and the polyvinyl alcohol is PVA1788.

[0022] Preferably, when preparing the W1 / O / W2 emulsion, the volume ratio of the internal aqueous phase, the oil phase and the polyvinyl alcohol aqueous solution is (0.9-1.2):5:(45-55).

[0023] In the preparation of the microcapsules of the present application, the ratio of the oil phase to the external aqueous phase is also one of the factors affecting the morphology and release rate of the microcapsules. Different ratios of the oil phase to the external aqueous phase have a greater impact on the appearance of the microcapsules after freeze-drying. If the amount of the oil phase and the polyvinyl alcohol aqueous solution is too large, the core material may not be fully wrapped by the wall material, and the raw drug may be exposed on the surface of the microcapsules, and the microcapsules may be in a lump shape. This is mainly because when the volume of the oil phase and the external aqueous phase polyvinyl alcohol aqueous solution increases, the suspension system will be unstable, and the pyraclostrobin with high density may settle, resulting in that the pyraclostrobin is not completely coated and exposed on the surface. In addition, if the amount of the polyvinyl alcohol aqueous solution is too large, the residual excess PVA may adhere to the drug-loaded microcapsules during the separation of the microcapsules due to the certain adhesive force of PVA, resulting in the agglomeration of the microcapsules.

[0024] Preferably, in step (3), the power of ultrasonic dispersion is 90-100 W; and in step (4), the power of ultrasonic dispersion is 190-200 W.

[0025] Preferably, in step (5), the temperature of rotary evaporation is 39-42℃; the centrifugal speed is 8000-15000 r / min, the centrifugal time is 8-15 min, and the number of centrifugal water washing is 2-4 times; after pre-freezing, the pyraclostrobin and brassinolide double-loaded microcapsules are obtained by vacuum freeze-drying in a freeze-drying machine.

[0026] In the present application, the organic solvent is removed by rotary evaporation, and the evaporation speed of the organic solvent is relatively fast by combining with a suitable rotary evaporation temperature. Therefore, the surface of the microcapsules will be quickly solidified, so that the encapsulation efficiency is improved, the burst release is reduced, and the release rate is slowed down.

[0027] Further preferably, in step (5), the temperature of rotary evaporation is 40℃; the centrifugal speed is 10000 r / min, the centrifugal time is 10 min, and the number of centrifugal water washing is 3 times; and the pre-freezing temperature is-20℃.

[0028] Further preferably, after the centrifugal water washing is completed, the container (centrifugal tube, etc.) containing the material obtained by centrifugal water washing can be wrapped with tin foil paper, so as to further prevent the photodegradation of the pyraclostrobin raw drug.

[0029] As a general inventive concept, the present application provides a use of the above-mentioned double-loaded microcapsules or the double-loaded microcapsules prepared by the above-mentioned preparation method in the cultivation of camellia oleifera fruits, atractylodes rhizome, polygonatum sibiricum or rice.

[0030] Compared with the prior art, the present application has the following beneficial effects:

[0031] 1、The present application adopts polylactic acid-glycolic acid copolymer (PLGA) and hydroxypropyl methyl cellulose phthalate (HP55) with a suitable mass ratio to cooperate as the capsule wall material, which can increase the drug loading capacity of the microcapsule and the drug encapsulation rate, control the drug diffusion rate, has good sustained-release effect, and the dual-loading microcapsule particle size is suitable, so that the dual-loading microcapsule has a long effective period, and the preparation method is simple, so that the prepared dual-loading microcapsule has uniform particle size distribution, no damage and no coating phenomenon.

[0032] 2、Since pyraclostrobin is prone to hydrolysis and photolysis, the preparation is prone to physical instability, and has high toxicity to aquatic organisms, which limits its application. The microencapsulation of pyraclostrobin can improve the environmental safety and persistence of pyraclostrobin. The present application combines 24-epibrassilicin lactone and pyraclostrobin, uses suitable wall materials and optimizes the preparation method, so that the synergistic regulation effect of 24-epibrassilicin lactone and pyraclostrobin can be efficiently exerted. The 24-epibrassilicin lactone can promote pesticide degradation, effectively prevent and alleviate the phytotoxicity caused by improper use of pyraclostrobin, and enhance the disease resistance of plants.

[0033] 3、The dual-loading microcapsule of the present application does not contain organic solvents, which can significantly reduce the environmental pollution caused by pesticides. The present application can solve the problems of poor stability, low utilization rate and short effective period of traditional preparations, and the 24-epibrassilicin lactone in the dual-loading microcapsule can promote crop growth at low concentrations, and the outer wall material can significantly improve the safety of pyraclostrobin preparation to aquatic organisms.

[0034] 4、The dual-loading microcapsule of the present application is suitable for oil tea fruit planting, atractylodes rhizome planting or polygonatum planting, has a long effective period and good control effect. BRIEF DESCRIPTION OF DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0036] Figure 1 The scanning electron microscope images of the dual-loading microcapsule prepared in Comparative Example 1 (a) and the dual-loading microcapsule prepared in Example 1 (b);

[0037] Figure 2Scanning electron microscope images of the dual-loaded microcapsules prepared in Example 1 (a) and the dual-loaded microcapsules prepared in Comparative Example 2 (b);

[0038] Figure 3 Particle size distribution graph of the dual-loaded microcapsules prepared in Example 1;

[0039] Figure 4 Release curve graph of the pyraclostrobin in the dual-loaded microcapsules (a) and the release curve graph of the brassinolide in the dual-loaded microcapsules (b);

[0040] Figure 5 Infrared spectrogram of the dual-loaded microcapsules prepared in Example 1; in the graph, (a) is the infrared spectrogram of the empty microcapsules; (b) is the infrared spectrogram of the dual-loaded microcapsules; (c) is the infrared spectrogram of the pyraclostrobin; (d) is the infrared spectrogram of the 24-epibrassinolide;

[0041] Figure 6 Thermogravimetric curve graph of the dual-loaded microcapsules prepared in Example 1; in the graph, (a) is the TG curve graph; (b) is the DTG curve graph;

[0042] Figure 7 Light stability test graph of the dual-loaded microcapsules prepared in Example 1;

[0043] Figure 8 Preparation process and effect graph of the dual-loaded microcapsules in Example 1. DETAILED DESCRIPTION

[0044] In order to facilitate the understanding of the present application, the following will be combined with the description of the preferred embodiments of the present application and the drawings to make a more comprehensive and detailed description of the present application, but the protection scope of the present application is not limited to the following specific embodiments.

[0045] Unless otherwise defined, all the professional terms used in the following have the same meaning as that generally understood by the person skilled in the art. The professional terms used in the present application are only for the purpose of describing the specific embodiments and are not intended to limit the protection scope of the present application.

[0046] Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in the present application can be purchased from the market or can be prepared by the existing method, as shown in Table 1.

[0047] Table 1:

[0048]

[0049] The preparation method of 1% w / v polyvinyl alcohol (PVA) aqueous solution in the following examples and comparative examples of the present application is as follows: 5 g of PVA 1788 is weighed and added to 500 mL of distilled water. The water bath is heated and slowly warmed to 90°C, and stirred at 500 r / min until the PVA is completely dissolved. After the PVA solution is cooled to room temperature, it is used again.

[0050] Example 1:

[0051] A kind of double-loading microcapsule containing pyraclostrobin and brassinolide, the double-loading microcapsule includes core material and wall material, and the wall material covers the core material;The core material includes pyraclostrobin and 24-epibrassinolide, and the wall material includes polylactic acid-hydroxyacetic acid copolymer and hydroxypropyl methyl cellulose phthalate.

[0052] As shown in Figure 8 , a preparation method of a double-loading microcapsule containing pyraclostrobin and brassinolide, comprising the following steps:

[0053] (1) Preparation of internal aqueous phase: 7 mg of 24-epibrassinolide technical material is weighed into a 10 mL centrifuge tube, 5 mL of acetone and 2 mL of distilled water are added, and ultrasonic treatment is performed to completely dissolve the mixture to obtain an internal aqueous phase.

[0054] (2) Preparation of oil phase: 46 mg of polylactic acid-hydroxyacetic acid copolymer (PLGA) is weighed into a 10 mL centrifuge tube, and 4 mL of dichloromethane is added, and ultrasonic treatment is performed to completely dissolve the mixture to obtain a PLGA solution;94 mg of hydroxypropyl methyl cellulose phthalate (HP55) is weighed into a 10 mL centrifuge tube, and 1 mL of acetone is added, and ultrasonic treatment is performed, then the above volume of PLGA solution is added, and ultrasonic treatment is continued to completely dissolve the HP55 to obtain a wall material solution;120 mg of pyraclostrobin is added to the obtained wall material solution, and ultrasonic treatment is performed to completely dissolve the pyraclostrobin to obtain an oil phase.

[0055] (3) 1 mL of the internal aqueous phase obtained in step (1) is slowly added to 5 mL of the oil phase obtained in step (2), and ultrasonic dispersion is performed for 5 s each time, and then the mixture is allowed to stand for 10 s, and the total ultrasonic dispersion time is 3 min, to obtain a W1 / O emulsion, wherein the ultrasonic dispersion power is 100 W.

[0056] (4) The W1 / O emulsion is quickly poured into 50 mL of a 1% w / v polyvinyl alcohol aqueous solution (external aqueous phase), and ultrasonic dispersion is performed for 3 s each time, and then the mixture is allowed to stand for 3 s, and the total ultrasonic dispersion time is 6 min, to obtain a W1 / O / W2 emulsion, wherein the ultrasonic dispersion power is 195 W.

[0057] (5) The W1 / O / W2 emulsion was rotary evaporated at 40℃ for 30 min to remove the organic solvents (acetone and dichloromethane) to form a microcapsule emulsion; the microcapsule emulsion was centrifuged at 10000 r / min for 10 min, and the supernatant was discarded, and the microcapsules were washed with distilled water and centrifuged for 3 times; after the centrifugal washing was completed, the centrifugal tube was wrapped with tin foil paper to prevent the original drug of pyraclostrobin from being photodegraded; the wrapped centrifugal tube was pre-frozen at -20℃, and then was placed in a freeze dryer for vacuum freeze drying to obtain the microcapsules (pyraclostrobin and brassinolide double-loaded microcapsules).

[0058] Example 2:

[0059] A pyraclostrobin and brassinolide double-loaded microcapsule, the double-loaded microcapsule comprising a core material and a wall material, the wall material coating the core material; the core material comprising pyraclostrobin and 24-epibrassinolide, and the wall material comprising polylactic acid-glycolic acid copolymer and hydroxypropyl methyl cellulose phthalate.

[0060] The preparation method of the above-mentioned pyraclostrobin and brassinolide double-loaded microcapsule, comprising the following steps:

[0061] (1) Preparation of inner water phase: 35 mg of 24-epibrassinolide original drug was weighed into a 10 mL centrifugal tube, 5 mL of acetone and 2 mL of distilled water were added, and ultrasonic treatment was performed to completely dissolve the mixture to obtain an inner water phase.

[0062] (2) Preparation of oil phase: 50 mg of polylactic acid-glycolic acid copolymer (PLGA) was weighed into a 10 mL centrifugal tube and 4 mL of dichloromethane was added, and ultrasonic treatment was performed to completely dissolve the mixture to obtain a PLGA solution; 90 mg of hydroxypropyl methyl cellulose phthalate (HP55) was weighed into a 10 mL centrifugal tube and 1 mL of acetone was added, and ultrasonic treatment was performed, then the above-mentioned volume of PLGA solution was added, and ultrasonic treatment was continued to completely dissolve the HP55 to obtain a wall material solution; 120 mg of pyraclostrobin was added to the obtained wall material solution, and ultrasonic treatment was performed to completely dissolve the mixture to obtain an oil phase.

[0063] (3) 1 mL of the inner water phase obtained in step (1) was slowly added to 5 mL of the oil phase obtained in step (2), and ultrasonic dispersion was performed for 5 s each time, and the mixture was allowed to stand for 10 s, and the total ultrasonic dispersion time was 3 min to obtain a W1 / O emulsion, wherein the ultrasonic dispersion power was 100 W.

[0064] (4) The W1 / O emulsion was quickly poured into 50 mL of a 1% w / v polyvinyl alcohol aqueous solution, and ultrasonic dispersion was performed for 3 s each time, and the mixture was allowed to stand for 3 s, and the total ultrasonic dispersion time was 6 min to obtain a W1 / O / W2 emulsion, wherein the ultrasonic dispersion power was 195 W.

[0065] (5) The W1 / O / W2 emulsion was rotary evaporated at 42℃ for 30 min to remove the organic solvents (acetone and dichloromethane) to form a microcapsule emulsion; the microcapsule emulsion was centrifuged at 12000 r / min for 8 min, and the supernatant was discarded, and the microcapsules were washed with distilled water and centrifuged for 3 times; after the centrifugal washing, the centrifugal tube was wrapped with tin foil paper to prevent the original drug of pyraclostrobin from photodegradation; the wrapped centrifugal tube was pre-frozen at -20℃, and then was placed in a freeze dryer for vacuum freeze drying to obtain the microcapsules (pyraclostrobin and brassinolide double-loaded microcapsules).

[0066] Example 3:

[0067] A pyraclostrobin and brassinolide double-loaded microcapsule, the double-loaded microcapsule comprising a core material and a wall material, the wall material coating the core material; the core material comprising pyraclostrobin and 24-epibrassinolide, and the wall material comprising polylactic acid-glycolic acid copolymer and hydroxypropyl methyl cellulose phthalate.

[0068] The preparation method of the above-mentioned pyraclostrobin and brassinolide double-loaded microcapsule, comprising the following steps:

[0069] (1) Preparation of inner water phase: 70 mg of 24-epibrassinolide original drug was weighed into a 10 mL centrifugal tube, 4 mL of acetone and 3 mL of distilled water were added, and ultrasonic treatment was performed to completely dissolve the mixture to obtain an inner water phase.

[0070] (2) Preparation of oil phase: 40 mg of polylactic acid-glycolic acid copolymer (PLGA) was weighed into a 10 mL centrifugal tube and 4 mL of dichloromethane was added, and ultrasonic treatment was performed to completely dissolve the mixture to obtain a PLGA solution; 100 mg of hydroxypropyl methyl cellulose phthalate (HP55) was weighed into a 10 mL centrifugal tube and 1 mL of acetone was added, and ultrasonic treatment was performed, then the above-mentioned volume of PLGA solution was added, and ultrasonic treatment was continued to completely dissolve the HP55 to obtain a wall material solution; 240 mg of pyraclostrobin was added to the obtained wall material solution, and ultrasonic treatment was performed to completely dissolve the mixture to obtain an oil phase.

[0071] (3) 1 mL of the inner water phase obtained in step (1) was slowly added to 5 mL of the oil phase obtained in step (2), and ultrasonic dispersion was performed for 5 s each time, and the mixture was allowed to stand for 10 s, and the total ultrasonic dispersion time was 3 min to obtain a W1 / O emulsion, wherein the ultrasonic dispersion power was 100 W.

[0072] (4) The W1 / O emulsion was quickly poured into 50 mL of a 1% w / v polyvinyl alcohol aqueous solution, and ultrasonic dispersion was performed for 3 s each time, and the mixture was allowed to stand for 3 s, and the total ultrasonic dispersion time was 6 min to obtain a W1 / O / W2 emulsion, wherein the ultrasonic dispersion power was 195 W.

[0073] (5) The W1 / O / W2 emulsion was rotary evaporated at 40℃ for 30 min to remove the organic solvents (acetone and dichloromethane) to form a microcapsule emulsion; the microcapsule emulsion was centrifuged at 10000 r / min for 10 min, the supernatant was discarded, and the microcapsules were washed with distilled water for 4 times by centrifugation; after the centrifugal washing was completed, the centrifuge tube was wrapped with tin foil paper to prevent the original pyraclostrobin from photodegradation; the wrapped centrifuge tube was pre-frozen at -20℃, and then was placed in a freeze dryer for vacuum freeze drying to obtain the microcapsules (pyraclostrobin and brassinolide double-loaded microcapsules).

[0074] Comparative Example 1

[0075] A preparation method of pyraclostrobin and brassinolide double-loaded microcapsules, which is different from Example 1 in that step (2) comprises the following steps: 60 mg of polylactic acid-glycolic acid copolymer (PLGA) was weighed into a 10 mL centrifuge tube, and 4 mL of dichloromethane was added, and the PLGA was completely dissolved by ultrasonic treatment to obtain a PLGA solution; 80 mg of hydroxypropyl methylcellulose phthalate (HP55) was weighed into a 10 mL centrifuge tube and 1 mL of acetone was added, and after ultrasonic treatment, the above volume of PLGA solution was added, and the HP55 was completely dissolved by continuing ultrasonic treatment to obtain a wall material solution; 120 mg of pyraclostrobin was added to the obtained wall material solution, and the pyraclostrobin was completely dissolved by ultrasonic treatment to obtain an oil phase.

[0076] Comparative Example 2

[0077] A preparation method of pyraclostrobin and brassinolide double-loaded microcapsules, which is different from Example 1 in that,

[0078] Step (3) comprises the following steps: 1 mL of the inner water phase obtained by step (1) was slowly added to 5 mL of the oil phase obtained by step (2), and ultrasonic dispersion was performed for 3 min to obtain a W1 / O emulsion, wherein the ultrasonic dispersion power was 100 W.

[0079] Step (4) comprises the following steps: the W1 / O emulsion was quickly poured into 50 mL of a 1% w / v polyvinyl alcohol aqueous solution, and ultrasonic dispersion was performed for 6 min to obtain a W1 / O / W2 emulsion, wherein the ultrasonic dispersion power was 195 W.

[0080] Performance test:

[0081] 1. Effect of mass ratio of polylactic acid-glycolic acid copolymer to hydroxypropyl methylcellulose phthalate in the wall material on the appearance of the microcapsules

[0082] From Figure 1-a) can be known, in the comparative example 1, when the mass ratio of the added polylactic acid-glycolic acid copolymer and hydroxypropyl methyl cellulose phthalate is 60:80, the final obtained double-loaded microcapsules are smooth and spherical, but breakage phenomenon occurs.

[0083] By Figure 1 -b) can be known, in the example 1 of the present application, when the mass ratio of the added polylactic acid-glycolic acid copolymer and hydroxypropyl methyl cellulose phthalate is 46:94, the final obtained double-loaded microcapsules have uniform particle size distribution and no breakage phenomenon.

[0084] 2, Effect of ultrasonic dispersion process on the appearance of microcapsules

[0085] By Figure 2 It can be known that the scanning electron microscope graph of the double-loaded microcapsules in the example 1 ( Figure 2 -a) and the scanning electron microscope graph of the double-loaded microcapsules in the comparative example 2 ( Figure 2 -b) can be known, although both of them are spherical, but in the comparative example 2, when preparing W1 / O / W2 emulsion, continuous ultrasonic dispersion is carried out without interval time, and the obtained microcapsules have more obvious adhesion phenomenon.

[0086] 3, Particle size and distribution of microcapsules

[0087] The surface morphology and overall distribution of the double-loaded microcapsules were observed by SEM (Scanning Electron Microscopy). The particle size of the microcapsules was calculated according to the scanning electron microscope graph using Image software.

[0088] By Figure 3 It can be known that the particle size and distribution of the microcapsules prepared in the example 1, the average particle size of the microcapsules is 2±1.2 μm, and the particle size is normally distributed. It can be seen that the double-loaded microcapsules of pyraclostrobin and brassinolide prepared by the preparation method of the present application have high stability, good film forming property, and can form microcapsules with small particle size.

[0089] 4, Encapsulation efficiency and drug loading of microcapsules

[0090] 5mg of double-loaded microcapsule sample in the example 1 was accurately weighed, dissolved by ultrasonic breaking of the wall with 10mL of methanol, and then a certain volume of 100mg / L phenylboronic acid solution was added, and a derivatization reaction was carried out in a water bath at 80℃. After the solution was evaporated to dryness, 10mL of methanol was added for re-dissolution, and the peak area of the effective ingredient in the microcapsules was determined by high performance liquid chromatography. The concentration of the effective ingredient was calculated by substituting into the standard curve. The drug loading and encapsulation efficiency of the double-loaded microcapsules were calculated by the following formula (1) and formula (2), respectively:

[0091]

[0092]

[0093] The encapsulation efficiency of the pyraclostrobin in the double-loaded microcapsules in Example 1 was 78.58% and the drug loading was 31.43% as measured by high performance liquid chromatography. The encapsulation efficiency of 24-epibrassilicin was 61.13% and the drug loading was 4.08%. The low drug loading of 24-epibrassilicin was probably caused by the low amount of 24-epibrassilicin used.

[0094] 5. Evaluation of the release performance of the microcapsules

[0095] In this test, the double-loaded microcapsules were directly put into the release medium, and the effective components in the microcapsules slowly diffused through the pores in the capsule wall. The release medium was then taken at certain time intervals, and finally, the release performance of the microcapsules was determined by high performance liquid chromatography.

[0096] The release curve of the double-loaded microcapsules is shown in Figure 4 As can be seen from the figure, the release amount of pyraclostrobin reached a maximum of 82.67% at 72h, and the release amount of 24-epibrassilicin reached a maximum of 65.08% at 48h. Pyraclostrobin and brassinolide were almost simultaneously released, which met the purpose of synergistic effect and exerted the drug efficacy at the same time.

[0097] 6. Infrared spectrum analysis of the microcapsules

[0098] An appropriate amount of KBr was mixed with the freeze-dried pyraclostrobin technical material, 24-epibrassilicin technical material, empty microcapsules, and double-loaded microcapsules in Example 1, respectively, and then tableted. The samples were analyzed and determined by FT-IR (Fourier Transform Infrared spectrometer).

[0099] As can be seen from b and c in Figure 5 , the absorption peaks at 1546cm -1 and 935cm -1 are the stretching vibration peaks of amide II band and -CH3 on the pyrazole ring, respectively, which are both characteristic absorption peaks of pyraclostrobin. The characteristic absorption peaks of pyraclostrobin were found in the double-loaded microcapsule sample (microcapsules in Example 1), indicating that pyraclostrobin was well coated in the wall material. Figure 5 As can be seen from d in Figure 5 , the absorption peaks at 3406cm -1 , 2959cm -1The functional groups represented by the absorption peaks of a, b and d are -OH and -CH3, respectively, which are characteristic absorption peaks of 24-epibrassilicin. Similar absorption peaks also appeared in the double-loaded microcapsule sample and the empty microcapsule, but comparison of a, b and d showed that the absorption peaks at these positions were strengthened after 24-epibrassilicin was coated, indicating that 24-epibrassilicin reacted with the wall material, possibly due to the interaction between the active ingredient and the wall material. These analysis results show that pyraclostrobin and 24-epibrassilicin were successfully coated in the microcapsules.

[0100] 7. Thermal gravimetric analysis of the microcapsules

[0101] Thermal gravimetric analysis method: The amount of sample added each time was 5-10 mg, and the temperature was raised at a rate of 20°C / min under the protection of nitrogen as the protective gas, and the temperature range was 50-600°C.

[0102] The thermal stability of the double-loaded microcapsules of the present application can be determined by thermal gravimetric analysis. From the TG curve and the DTG curve of the microcapsules, Figure 6 it can be seen that the decomposition temperature of pyraclostrobin is 200°C, the temperature at which complete decomposition occurs is about 450°C, and the mass loss is 80%. 24-epibrassilicin has a small amount of weight loss at 40-100°C due to the evaporation of water contained in the active ingredient, and is thermally stable at 100-310°C with almost no weight loss. The decomposition temperature of 24-epibrassilicin is about 310°C, and the temperature at which complete decomposition occurs is 460°C. At 210°C, the double-loaded microcapsules (microcapsules in Example 1) exhibit the decomposition of pyraclostrobin, and when pyraclostrobin is not completely decomposed, the decomposition of 24-epibrassilicin also occurs. When the temperature reaches 450°C, both active ingredients have completely decomposed. This fully demonstrates that the coating of the wall material improves the thermal stability of the core material to a certain extent.

[0103] 8. Evaluation of the light stability of the microcapsules

[0104] The 5 mg double-loaded microcapsules were dispersed and suspended in 10 mL of methanol by ultrasonic cell pulverizer, and the initial concentration of the pyraclostrobin was 220 mg / L, and the 24-EBL (24-epibrassinolid) was 2 mg / L. At the same time, a control group was set up, and the same concentration of pyraclostrobin and 24-EBL technical were prepared as a control. 3 mL of the solution was added to each 10 mL centrifuge tube, and then placed under a 500 W ultraviolet high-pressure mercury lamp, with a distance of 30 cm from the lamp to the liquid level. The experiment adopted an independent parallel experiment method. A total of 10 centrifuge tubes were taken out at each time interval. The solution in the centrifuge tube was filtered to 2 mL, and the pyraclostrobin technical solution could be directly detected after filtering the membrane, and the double-loaded microcapsule solution and the 24-EBL technical solution needed to be derivatized by phenylboronic acid before detection, and then concentrated by 10 times before being detected by HPLC. 2 mL of the solution was taken in a centrifuge tube, a certain amount of phenylboronic acid was added, and heated in a 80° water bath until the methanol was completely evaporated, and then dissolved with 0.2 mL of methanol, and filtered for detection.

[0105] Through the photodegradation experiment, as shown in Figure 7 It can be seen that the photolysis efficiency of pyraclostrobin and 24-EBL in the double-loaded microcapsule is obviously lower than that of the technical, which shows that the wall material has a certain protective effect on the active ingredients. Since the pyraclostrobin in the double-loaded microcapsule is wrapped in the outer layer, the degradation rate does not increase obviously, while the 24-EBL in the inner layer can significantly improve the light stability. The degradation rate of 24-EBL increases rapidly with time, and only 8.31% remains after 20 min. The degradation rate of 24-EBL in the double-loaded microcapsule increases slowly, and is still 60.6% after 20 min, and the half-life of light is 4.36 times that of 24-EBL material. This fully shows that the double-loaded microcapsule improves the light stability of the core material, which is beneficial to prolong the use time of the active ingredient and improve its utilization rate.

[0106] 9. Acute toxicity experiment of microcapsules on zebrafish

[0107] Pyraclostrobin has great toxicity to aquatic organisms, so the application of traditional formulations in rice fields is limited. In order to verify the toxicity of the double-loaded microcapsule to aquatic organisms, the present application selects zebrafish as a model organism. During the experiment, no zebrafish died in the blank control group. The experimental results are shown in Table 2, and the LC 50 (96h) of pyraclostrobin EC on zebrafish is 0.113 mg / L. Therefore, pyraclostrobin EC has acute toxicity to zebrafish. The LC 50 (96h) of the double-loaded microcapsule is 2.344 mg / L, and the toxicity of the double-loaded microcapsule to zebrafish is obviously lower than that of pyraclostrobin EC, which is reduced by 20 times. This fully shows that the shell material of the double-loaded microcapsule isolates pyraclostrobin from water, effectively reducing the toxicity of the technical to aquatic organisms.

[0108] Table 2:

[0109]

[0110] 10. Control effect of microcapsules on rice blast

[0111] The control effect of the emulsifiable concentrate prepared by the double-loaded microcapsule and the pyraclostrobin on rice blast was tested by pot experiment. The disease index and fresh weight growth rate were counted after 8 days. The results of pot experiment (Table 3) showed that the disease index gradually increased with the decrease of the concentration of pyraclostrobin emulsifiable concentrate. The same trend was also observed for the double-loaded microcapsule. The control effect of the double-loaded microcapsule was better than that of the pyraclostrobin emulsifiable concentrate at the same concentration. At the same time, the effect of the two preparations on the fresh weight of rice showed that the double-loaded microcapsule had a more significant effect on the fresh weight of the plant at low concentration than the emulsifiable concentrate, and was slightly inferior to the emulsifiable concentrate at high concentration. Moreover, the fresh weight efficiency percentage increased first and then decreased with the increase of the concentration of the preparation. The possible reason for this phenomenon was that the emulsifiable concentrate prepared by pyraclostrobin had strong quick-acting property, and the preparation itself had the effect of promoting plant growth, so the instantaneous effect of promoting plant growth was more obvious at high concentration. However, the double-loaded microcapsule preparation had excellent slow-release effect, effectively prolonged the effective period, and showed the advantages of microcapsule.

[0112] Table 3:

[0113]

Claims

1. A dual-loaded microcapsule containing pyraclostrobin and brassinolide, characterized in that, The double-loading microcapsule comprises a core material and a wall material, and the wall material covers the core material; the core material comprises pyraclostrobin and 24-epibrassinolide, and the wall material comprises polylactic acid-glycolic acid copolymer and hydroxypropyl methyl cellulose phthalate; The mass ratio of the polylactic acid-glycolic acid copolymer and the hydroxypropyl methyl cellulose phthalate is (40-50):(90-100); the number average molecular weight of the polylactic acid-glycolic acid copolymer is 9000-13000; The preparation method of the double-loading microcapsule containing pyraclostrobin and brassinolide comprises the following steps: (1) dissolving 24-epibrassinolide in a mixed solution of acetone and water to obtain an inner water phase; (2) dissolving polylactic acid-glycolic acid copolymer in dichloromethane to obtain a polylactic acid-glycolic acid copolymer solution; adding hydroxypropyl methyl cellulose phthalate into acetone, ultrasonic treatment, then adding the polylactic acid-glycolic acid copolymer solution and continuing ultrasonic treatment to obtain a wall material solution; adding pyraclostrobin into the wall material solution and ultrasonic treatment to obtain an oil phase; (3) slowly adding the inner water phase into the oil phase, ultrasonic dispersing for 4-5 s each time, standing for 9-12 s each time, and ultrasonic dispersing for a total of 3-4 min to obtain a W1 / O emulsion; (4) adding the W1 / O emulsion into a polyvinyl alcohol aqueous solution, ultrasonic dispersing for 3-4 s each time, standing for 3-4 s each time, and ultrasonic dispersing for a total of 5-7 min to obtain a W1 / O / W2 emulsion; When preparing the W1 / O / W2 emulsion, the volume ratio of the inner water phase, the oil phase and the polyvinyl alcohol aqueous solution is (0.9-1.2):5:(45-55); (5) performing rotary evaporation on the W1 / O / W2 emulsion to remove acetone and dichloromethane, obtaining a microcapsule emulsion; performing centrifugal water washing on the microcapsule emulsion, and then performing vacuum freeze drying to obtain the double-loading microcapsule containing pyraclostrobin and brassinolide.

2. The dual-load microcapsule of claim 1, wherein, The particle size of the double-loading microcapsule is 0.75-5.5 μm.

3. The dual-load microcapsule of claim 1, wherein In step (1), the mass-volume ratio of the brassinolide, acetone and water in the inner water phase is (7-70) mg:(4-5) mL:(2-3) mL; in step (2), the mass-volume ratio of pyraclostrobin, polylactic acid-glycolic acid copolymer, hydroxypropyl methyl cellulose phthalate, dichloromethane and acetone in the oil phase is (60-240) mg:(40-50) mg:(90-100) mg:4 mL:1 mL.

4. The dual-load microcapsule of claim 1, wherein, In step (4), the concentration of the polyvinyl alcohol aqueous solution is 1% w / v, and the polyvinyl alcohol is PVA1788.

5. The dual-carrier microcapsule according to any one of claims 1 to 4, characterized in that, In step (3), the ultrasonic dispersing power is 90-100 W; in step (4), the ultrasonic dispersing power is 190-200 W.

6. The dual-carrier microcapsule according to any one of claims 1 to 4, characterized in that, In step (5), the temperature of rotary evaporation is 39-42℃; the centrifugal speed is 8000-15000 r / min, the centrifugal time is 8-15 min, and the centrifugal water washing times are 2-4 times; after pre-freezing, the product is placed into a freeze dryer for vacuum freeze drying to obtain the pyraclostrobin and brassinolide double-loaded microcapsules.

7. The double-loaded microcapsules according to any one of claims 1-6 for use in oil tea fruit planting, atractylodes rhizome planting, polygonatum planting or rice planting.

Images