Preparation method of bovine serum albumin diflubenzuron particles

Abstract

The invention discloses a preparation method of bovine serum albumin chlorbenzuron particles, and relates to the technical field of pesticide preparation.According to the preparation method, chlorbenzuron is wrapped with bovine serum albumin, nanocrystallization treatment is achieved, the solubility, stability and bioavailability of chlorbenzuron are improved, the absorption effect of chlorbenzuron in pest bodies is enhanced, and the pesticide effect is improved; the preparation method comprises the following steps: dissolving bovine serum albumin in a water phase to prepare a bovine serum albumin solution; the method comprises the following steps: dissolving a chlorbenzuron active compound in an acetonitrile solution under a water bath heating condition to form a chlorbenzuron organic phase; under a stirring condition, dropwise adding a bovine serum albumin solution into the diflubenzuron organic phase, and continuously heating and stirring to enable the bovine serum albumin to wrap the diflubenzuron to form nanoparticles; the bovine serum albumin and chlorbenzuron nanoparticles are separated by adopting a centrifugal method and are freeze-dried to finally obtain the bovine serum albumin and chlorbenzuron nanoparticles, and the prepared nanoparticles are uniform in particle size and good in dispersity, have the controlled release characteristic and can effectively prolong the drug effect lasting time.

Description

Technical Field

[0001] The present invention relates to the technical field of pesticide preparation, and particularly to a method for preparing bovine serum albumin chlorbenzuron microparticles. Background Art

[0002] Chlorbenzuron belongs to benzoylurea insect chitin synthesis inhibitors and is an insect growth regulator; its mechanism of action is mainly to inhibit the activity of insect cuticle chitin synthase, affect the processes of insect molting and embryonic development, and cause pests to be unable to complete normal growth and development and die.

[0003] In addition, chlorbenzuron also has an inhibitory effect on the respiratory metabolism of pest eggs and the DNA and protein metabolism during embryonic development, resulting in a lack of chitin in the larvae in the eggs, unable to hatch or die quickly after hatching.

[0004] At present, the chlorbenzuron pesticides on the market are mainly in the form of microemulsion formulations, which mainly play a role after being ingested by pests. Although the microemulsion formulation of chlorbenzuron has played a certain role in the control of agricultural pests, the chlorbenzuron technical material is insoluble in water and only soluble in organic solvents, which makes it have certain limitations in field applications and requires the use of organic solvents. In addition, the existing microemulsion formulation of chlorbenzuron is easily degraded under light and high temperature conditions, resulting in a reduction in drug efficacy and affecting the actual control effect; and due to the relatively large particle size, chlorbenzuron mainly relies on being ingested by pests to enter the body and play a role. If pests only crawl in the application area but do not ingest, the control effect is limited. Summary of the Invention

[0005] In view of the above existing problems, the present invention is proposed.

[0006] The present invention provides a method for preparing bovine serum albumin chlorbenzuron microparticles to solve the problems of poor solubility, low photo-thermal stability and single mode of action of existing chlorbenzuron preparations.

[0007] To solve the above technical problems, the present invention provides the following technical solutions:

[0008] An embodiment of the present invention provides a method for preparing bovine serum albumin chlorbenzuron microparticles, which includes,

[0009] Step S1, dissolve bovine serum albumin in the aqueous phase, weigh 0.02 g to 0.16 g of bovine serum albumin, add 2 mL to 6 mL of tertiary water, and dissolve it under the condition of water bath heating at 50 °C to obtain an aqueous solution of bovine serum albumin;

[0010] Step S2, prepare the chlorbenzuron organic phase under the condition of water bath heating, weigh 0.08 g to 0.32 g of chlorbenzuron technical material, add 20 mL to 60 mL of acetonitrile solution, and stir at a speed of 800 rpm to 1000 rpm under the condition of water bath at 50 °C until the chlorbenzuron technical material is completely dissolved;

[0011] Step S3, under stirring conditions, an aqueous solution of bovine serum albumin is added dropwise to the chlorbenzuron organic phase, controlling the dropping rate. After the dropping is completed, continue to stir and heat in a water bath for 10 - 30 min to enable bovine serum albumin to encapsulate chlorbenzuron to form nanoparticles.

[0012] Step S4, the nanoparticles are separated by centrifugation, centrifuged at a speed of 4000 rpm to 6000 rpm for 5 - 15 min, the supernatant is discarded, the precipitate is retained, and freeze-dried for 24 h to 72 h to finally obtain bovine serum albumin-chlorbenzuron nanoparticles.

[0013] As a preferred embodiment of the preparation method of the bovine serum albumin-chlorbenzuron microparticles of the present invention, wherein: the bovine serum albumin is selected from biological sources or recombinant sources and has biocompatibility and biodegradability.

[0014] As a preferred embodiment of the preparation method of the bovine serum albumin-chlorbenzuron microparticles of the present invention, wherein: the solvent of the chlorbenzuron organic phase is acetonitrile or other organic solvents that can dissolve chlorbenzuron, such as acetone.

[0015] As a preferred embodiment of the preparation method of the bovine serum albumin-chlorbenzuron microparticles of the present invention, wherein: the dropping process is carried out by dropping at a constant rate to ensure the uniformity and dispersion of the nanoparticles.

[0016] As a preferred embodiment of the preparation method of the bovine serum albumin-chlorbenzuron microparticles of the present invention, wherein: the stirring speed during the stirring process is 800 rpm to 1000 rpm, and the optimal stirring speed is 900 rpm.

[0017] As a preferred embodiment of the preparation method of the bovine serum albumin-chlorbenzuron microparticles of the present invention, wherein: the centrifugation process uses a centrifugation speed of 5000 rpm and the centrifugation time is 10 min.

[0018] As a preferred embodiment of the preparation method of the bovine serum albumin-chlorbenzuron microparticles of the present invention, wherein: the time range of the freeze-drying step is 24 h to 72 h, and the optimal freeze-drying time is 48 h.

[0019] As a preferred embodiment of the preparation method of the bovine serum albumin-chlorbenzuron microparticles of the present invention, wherein: the particle size range of the nanoparticles is 50 nm to 500 nm, and the optimal particle size is controlled at 100 nm to 200 nm.

[0020] As a preferred embodiment of the preparation method of the bovine serum albumin-chlorbenzuron microparticles of the present invention, wherein: the chlorbenzuron can be replaced with other hydrophobic pesticides that are soluble in the organic phase solvent.

[0021] The present invention also provides a nano-pesticide bovine serum albumin chlorbenzuron particle. Based on the above preparation method of the bovine serum albumin chlorbenzuron particle, the particle is formed by wrapping chlorbenzuron with bovine serum albumin and is prepared through centrifugal separation and freeze-drying processes.

[0022] The beneficial effects of the present invention are as follows:

[0023] In the present invention, bovine serum albumin is used as a carrier to wrap chlorbenzuron, making the nano-particles easier to be swallowed and absorbed by pests, improving the utilization rate of chlorbenzuron, and enhancing the contact-killing and stomach toxicity effects.

[0024] The existing chlorbenzuron technical material is hardly soluble in water. The present invention improves its water dispersibility through nano-encapsulation technology, making it more efficient during field application and reducing the dependence on organic solvents.

[0025] The traditional chlorbenzuron microemulsion is easily degraded under light and high-temperature conditions, while the nano-particles prepared in the present invention have good stability and can still maintain a high pesticidal effect under environmental influences.

[0026] In the present invention, the nano-particle structure enables chlorbenzuron to be slowly released, prolonging the duration of the pesticidal effect, reducing the number of repeated applications, and thus reducing the cost of pesticide use.

[0027] In the present invention, due to the small size of the nano-particles, they can more easily penetrate the epidermis of pests, improving the control effect on non-feeding pests and expanding the applicable range of chlorbenzuron.

[0028] In the present invention, using bovine serum albumin as a carrier has good biocompatibility and degradability, is non-toxic and harmless, and at the same time reduces the use of organic solvents, conforming to the development trend of green agriculture.

[0029] In the present invention, the preparation method of the present invention is simple to operate, does not require special chemical reagents, does not require complex equipment, is suitable for large-scale production, can effectively reduce the production cost of pesticides, and improve the industrial application value.

[0030] In summary, the present invention optimizes the physical and chemical properties of chlorbenzuron through nano-encapsulation technology, improves the control effect and environmental friendliness of pesticides, and provides a new type of highly efficient, low-toxic and safe pesticide preparation for agricultural pest control. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0032] Figure 1 Field emission electron microscopy morphology diagram of the preparation method of the bovine serum albumin chlorbenzuron microparticles of the present invention;

[0033] Figure 2 Particle size distribution diagram of the preparation method of the bovine serum albumin chlorbenzuron microparticles of the present invention;

[0034] Figure 3 Drug loading rate diagrams with different ratios of the preparation method of the bovine serum albumin chlorbenzuron microparticles of the present invention;

[0035] Figure 4 Drug loading rate diagrams at different stirring speeds of the preparation method of the bovine serum albumin chlorbenzuron microparticles of the present invention;

[0036] Figure 5 Drug loading rate diagrams at different temperatures of the preparation method of the bovine serum albumin chlorbenzuron microparticles of the present invention. Specific embodiments

[0037] To make the above objects, features, and advantages of the present invention more obvious and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings of the specification.

[0038] Many specific details are set forth in the following description in order to fully understand the present invention, but the present invention may also be implemented in other ways different from those described herein. Those skilled in the art can make similar generalizations without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0039] Secondly, the so-called "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation manner of the present invention. The appearances of "in one embodiment" in different places in this specification do not all refer to the same embodiment, nor are they separate or alternative embodiments that exclude each other with other embodiments.

[0040] Bovine serum albumin (BSA) is a natural protein widely used in the biomedical field, with advantages such as good biocompatibility, non-toxicity, biodegradability, and non-immunogenicity; therefore, using bovine serum albumin as a carrier material to encapsulate chlorbenzuron to form nanoparticles can improve the drug efficacy, make it easier to be absorbed by pests, and at the same time have a controlled release effect, prolong the duration of drug efficacy, reduce the amount of pesticide used, and improve the utilization rate; in addition, the nanosized chlorbenzuron microparticles can increase the contact area between the drug and the target organisms, thereby enhancing the pest control effect and achieving the goals of high efficiency, low toxicity, and safety in prevention and control.

[0041] Without changing the structure of bovine serum albumin, the present invention utilizes its biocompatibility and nano - encapsulation characteristics to encapsulate chlorbenzuron in a nano - scale form. The present invention can not only improve the solubility and stability of chlorbenzuron, but also reduce the use of organic solvents, conforming to the development trend of green environmental protection agriculture. At the same time, it can improve the drug efficacy and enhance the pest control effect.

[0042] Example 1, referring to Figures 1-5 , this example provides a preparation method of bovine serum albumin and chlorbenzuron with different ratios. The specific steps include:

[0043] (1) Weigh 0.02 g, 0.04 g, 0.08 g, and 0.16 g of bovine serum albumin respectively and add them to 4 ml of tertiary water. Heat in a water bath at 50 °C to dissolve the bovine serum albumin powder and prepare an aqueous phase.

[0044] (2) Measure 40 mL of acetonitrile solution and place it in a small beaker. Heat in a water bath at 50 °C and stir the acetonitrile solution at a speed of 900 rpm simultaneously.

[0045] (3) Weigh 0.16 g of chlorbenzuron technical and add it to the acetonitrile solution. Continue to heat and stir the chlorbenzuron - acetonitrile solution under the above conditions until the chlorbenzuron technical is completely dissolved to form an oil phase.

[0046] (4) Continue to heat and stir the oil phase, and simultaneously add the aqueous solution of bovine serum albumin drop by drop. After the addition is complete, continue to heat and stir in a water bath for 15 - 25 min.

[0047] (5) After taking out the solution, centrifuge it at a speed of 5000 rpm for 10 min, discard the supernatant and retain the precipitate.

[0048] (6) Freeze - dry the above - obtained precipitate for 48 h to obtain a white powder, namely bovine serum albumin - chlorbenzuron nanoparticles.

[0049] The object of encapsulation by bovine serum albumin, chlorbenzuron, is a hydrophobic drug, and the method is also applicable to other hydrophobic drugs.

[0050] The acetonitrile solution in step (2) can be replaced with other organic solvents that can dissolve pesticide technicals, such as acetone, etc.

[0051] The chlorbenzuron in step (3) can also be replaced with other hydrophobic drugs that are soluble in organic solvents such as acetonitrile, and the encapsulation method is also applicable.

[0052] The stirring time in step (4) is 15 - 25 min, and the optimal condition is 20 min. The obtained nanoparticles have uniform particle size and good dispersibility.

[0053] Example 2, this example provides a preparation method of bovine serum albumin and chlorbenzuron with different stirring speeds. The specific steps include:

[0054] (1) Weigh 0.04 g of bovine serum albumin and add it to 4 ml of tertiary water. Heat it in a water bath at 50 °C to dissolve the bovine serum albumin powder and prepare the aqueous phase.

[0055] (2) Measure 40 mL of acetonitrile solution and place it in a small beaker. Heat it in a water bath at 50 °C and stir the acetonitrile solution at 800 rpm / 900 rpm / 1000 rpm respectively.

[0056] (3) Weigh 0.16 g of chlorbenzuron technical and add it to the acetonitrile solution. Continue to heat and stir the chlorbenzuron-acetonitrile solution under the above conditions until the chlorbenzuron technical is completely dissolved to obtain the oil phase.

[0057] (4) Continue to heat and stir the oil phase, and at the same time, gradually add the aqueous solution of bovine serum albumin drop by drop. After the addition is complete, continue to heat and stir in a water bath for 15 - 25 min.

[0058] (5) After taking out the solution, centrifuge it at 5000 rpm for 10 min, discard the supernatant and retain the precipitate.

[0059] (6) Freeze-dry the precipitate obtained above for 48 h to obtain a white powder, namely bovine serum albumin-chlorbenzuron nanoparticles.

[0060] Example 3. This example provides a preparation method of bovine serum albumin and chlorbenzuron at different temperatures. The specific steps are as follows:

[0061] (1) Weigh 0.04 g of bovine serum albumin and add it to 4 ml of tertiary water. Heat it in a water bath at 50 °C to dissolve the bovine serum albumin powder and prepare the aqueous phase.

[0062] (2) Measure 40 mL of acetonitrile solution and place it in a small beaker. Heat it in a water bath at 40 °C / 50 °C / 60 °C and stir the acetonitrile solution at 900 rpm.

[0063] (3) Weigh 0.16 g of chlorbenzuron technical and add it to the acetonitrile solution. Continue to heat and stir the chlorbenzuron-acetonitrile solution under the above conditions until the chlorbenzuron technical is completely dissolved to obtain the oil phase.

[0064] (4) Continue to heat and stir the oil phase, and at the same time, gradually add the aqueous solution of bovine serum albumin drop by drop. After the addition is complete, continue to heat and stir in a water bath for 15 - 25 min.

[0065] (5) After taking out the solution, centrifuge it at 5000 rpm for 10 min, discard the supernatant and retain the precipitate.

[0066] (6) Freeze-dry the precipitate obtained above for 48 h to obtain a white powder, namely bovine serum albumin-chlorbenzuron nanoparticles.

[0067] Example 3 provides a preparation method of standard bovine serum albumin and chlorbenzuron, and the specific steps are as follows:

[0068] (1) Weigh 0.04 g of bovine serum albumin and add it to 4 ml of tertiary water. Heat it in a water bath at 50 °C to dissolve the bovine serum albumin powder and prepare an aqueous phase.

[0069] (2) Measure 40 mL of acetonitrile solution and place it in a small beaker. Heat it in a water bath at 50 °C and stir the acetonitrile solution at a speed of 900 rpm simultaneously.

[0070] (3) Weigh 0.16 g of chlorbenzuron technical and add it to the acetonitrile solution. Continue to heat and stir the chlorbenzuron-acetonitrile solution under the above conditions until the chlorbenzuron technical is completely dissolved to obtain an oil phase.

[0071] (4) Continue to heat and stir the oil phase, and simultaneously add the aqueous solution of bovine serum albumin drop by drop. After the addition is complete, continue to heat and stir in a water bath for 15 - 25 min.

[0072] (5) After taking out the solution, centrifuge it at a speed of 5000 rpm for 10 min, discard the supernatant and retain the precipitate.

[0073] (6) Freeze-dry the obtained precipitate for 48 h to obtain a white powder, namely bovine serum albumin-chlorbenzuron nanoparticles.

[0074] The chlorbenzuron encapsulated by bovine serum albumin is a hydrophobic drug and is also applicable to other hydrophobic drugs.

[0075] The acetonitrile solution in step (2) can be replaced by other organic solvents that can dissolve pesticide technicals, such as acetone, etc.

[0076] The chlorbenzuron in step (3) can also be replaced by other hydrophobic drugs that are soluble in organic solvents such as acetonitrile, and its encapsulation method is also applicable.

[0077] The stirring time in step (4) is 15 - 25 min, and the optimal condition is 20 min. The obtained nanoparticles have uniform particle size and good dispersibility.

[0078] All the acetonitrile solutions used in the preparation process are of analytical purity, and all the aqueous solutions are of tertiary water. The technical features of the present invention are not limited to this, and any other embodiments obtained by those skilled in the art without departing from the technical solution of the present invention should be covered within the patent scope of the present invention.

[0079] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing bovine serum albumin benzyl alcohol microparticles, characterized in that: include, Step S1, dissolving bovine serum albumin in an aqueous phase, weighing 0.02 g to 0.16 g of bovine serum albumin, adding 2 mL to 6 mL of grade tertiary water, and dissolving in a 50° C. water bath to obtain a bovine serum albumin aqueous solution; Step S2, under a water bath heating condition, preparing a diflubenzuron organic phase, weighing 0.08 g to 0.32 g of diflubenzuron technical drug, adding 20 mL to 60 mL of acetonitrile solution, and stirring at a speed of 800 rpm to 1000 rpm under a 50° C. water bath condition until the diflubenzuron technical drug is completely dissolved; Step S3, under stirring conditions, adding a bovine serum albumin aqueous solution to the diflubenzuron organic phase dropwise, controlling the dropping rate, and continuing to heat and stir in a water bath for 10-30 minutes after the dropwise addition is completed, so that the bovine serum albumin encapsulates the diflubenzuron to form nanoparticles; Step S4, separating the nanoparticles by centrifugation at 4000 rpm to 6000 rpm for 5-15 min, discarding the supernatant, retaining the precipitate, and freeze-drying for 24 h to 72 h, finally obtaining bovine serum albumin-bifenazolamide nanoparticles.

2. The method for preparing bovine serum albumin-benzyl urea microparticles according to claim 1, characterized in that: The bovine serum albumin is selected from biological sources or recombinant sources and has biocompatibility and degradability.

3. The method for preparing bovine serum albumin-benzyl urea microparticles according to claim 2, characterized in that: The solvent of the diflubenzuron organic phase is acetonitrile or other organic solvents that can dissolve diflubenzuron.

4. The method for preparing bovine serum albumin benzyl alcohol microparticles according to claim 3, characterized in that: The dropping process adopts a constant rate dropwise adding method.

5. The method for preparing bovine serum albumin-benzyl urea microparticles according to claim 4, characterized in that: The stirring speed of the stirring process is 800 rpm to 1000 rpm, and the optimal speed is 900 rpm.

6. The method for preparing bovine serum albumin-benzyl urea microparticles according to claim 5, characterized in that: The centrifugal process adopts a centrifugal speed of 5000 rpm and a centrifugal time of 10 minutes.

7. The method for preparing bovine serum albumin-benzyl urea microparticles according to claim 6, characterized in that: The time range of the freeze-drying step is 24h to 72h.

8. The method for preparing bovine serum albumin-benzyl urea microparticles according to claim 7, characterized in that: The particle size of the nanoparticles ranges from 50 nm to 500 nm.

9. The method for preparing bovine serum albumin-benzyl urea microparticles according to claim 8, characterized in that: The diflubenzuron can be replaced by other hydrophobic pesticides that are soluble in the organic phase solvent.

10. A nano pesticide bovine serum albumin diflubenzuron microparticle, based on the preparation method of the bovine serum albumin diflubenzuron microparticle according to any one of claims 1 to 9, characterized in that: The microparticles are formed by encapsulating diflubenzuron with bovine serum albumin and are prepared by centrifugation and freeze-drying processes.

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