Matrine slow-release agent as well as preparation method and application thereof

Abstract

The invention discloses a matrine slow-release agent as well as a preparation method and application thereof, and belongs to the technical field of pesticides. The preparation method comprises the following steps: firstly, dissolving matrine in an acidic aqueous solution, then sequentially adding carboxyl cellulose and fatty amine into the solution, stirring and emulsifying to obtain a dispersion liquid, and finally, adding the dispersion liquid into vegetable oil, and carrying out secondary emulsification to obtain the W / O / W Pickering emulsion. The sophocarpidine is combined and self-assembled through non-covalent bonds to form the drug-loaded gel, a wrapping structure is formed, the stability of the drug-loaded gel is effectively improved, and meanwhile the slow release performance of drug molecules is achieved through the non-covalent bond combination form; and vegetable oil is used for encapsulation, so that the stability of matrine molecules is further ensured, the matrine has better affinity with the plant surface, and the wettability is improved.

Description

Technical Field

[0001] This invention belongs to the field of pesticide technology, specifically relating to a matrine sustained-release agent, its preparation method, and its application. Background Technology

[0002] Matrine, a quinolone alkaloid extracted from the legume *Sophora flavescens*, possesses broad-spectrum insecticidal, antibacterial, and antiviral activities and has been used as a main component in plant-derived pesticides for agricultural pest and disease control. Recent studies have found that matrine not only possesses biological activity itself, but its polar groups (such as hydroxyl and amino groups) in its molecular structure also endow it with excellent emulsifying, dispersing, and adhesive properties. It can form hydrogen bonds or van der Waals forces with pesticide active ingredients, improving the physicochemical properties of formulations. Furthermore, matrine can activate the plant immune system, synergistically enhancing the effects of insecticidal / fungal agents. Its natural origin also significantly reduces the toxicity risk of traditional adjuvants to non-target organisms. Therefore, developing matrine into a multifunctional pesticide adjuvant can improve pesticide utilization, reduce chemical pesticide usage, and align with the green plant protection concept of "reduced application and increased efficiency," making it of significant application value.

[0003] Current matrine formulations are not conducive to target absorption and improving control efficacy. They fail to address the poor stability and short half-life of matrine. Traditional matrine emulsifiable concentrate formulations require the addition of large amounts of environmentally polluting and harmful organic solvents during preparation. The most effective application method for controlling leek maggots using aqueous solutions and emulsifiable concentrates is root irrigation, which involves multiple applications and a large workload, making it unsuitable for the current large-scale industrialization of agriculture. Therefore, there is an urgent need to develop a preparation method for matrine formulations with good stability, high foliar deposition efficiency, and a long release period. Summary of the Invention

[0004] The first technical problem to be solved by this invention is to provide a matrine sustained-release agent with good stability, high leaf deposition efficiency, and long release period. The second technical problem to be solved by this invention is to provide a method for preparing the matrine sustained-release agent, which improves its stability by forming a drug-loaded gel through non-covalent self-assembly of matrine, thus creating an encapsulation structure. The third technical problem to be solved by this invention is to provide the application of this sustained-release agent in pesticide adjuvants.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] A method for preparing a matrine sustained-release agent involves first dissolving matrine in an acidic aqueous solution, then sequentially adding carboxycellulose and fatty amine to the solution and stirring and emulsifying to obtain a dispersion, and finally adding the dispersion to vegetable oil for secondary emulsification to obtain a W / O / W Pickering emulsion.

[0007] Furthermore, the pH of the acidic solution is 5.5 to 7.4.

[0008] Furthermore, the mass ratio of matrine, carboxycellulose, and fatty amine is 3:1:2 to 3:2:3.

[0009] Furthermore, the fatty amine has 12 to 18 carbon atoms, and the amino group is located at position 1.

[0010] Furthermore, the molecular weight of the carboxycellulose is 12,000 to 15,000.

[0011] Furthermore, the emulsification temperature is 25–30°C, and the time is 10 min.

[0012] Furthermore, the volume ratio of the dispersion to the vegetable oil is 5 to 6:1.

[0013] Furthermore, the secondary emulsification is carried out at a temperature of 25–30°C for 10 minutes.

[0014] Furthermore, the method for preparing the matrine sustained-release agent yields the matrine sustained-release agent.

[0015] Furthermore, the application of the matrine sustained-release agent in pesticide adjuvants.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] (1) This invention forms a drug-loaded gel by self-assembling matrine with non-covalent bonds, thereby forming an encapsulation structure that effectively improves its stability. At the same time, the non-covalent bond form enables sustained release of drug molecules.

[0018] (2) This invention utilizes vegetable oil encapsulation, which further ensures the stability of matrine molecules and provides better affinity and wettability to plant surfaces. Simultaneously, the prepared matrine formulation exhibits good stability, high leaf deposition efficiency, and a long release period. Attached Figure Description

[0019] Figure 1 A process flow diagram for preparing matrine sustained-release agent according to this application;

[0020] Figure 2 This is a fluorescence inverted microscope image of the matrine sustained-release emulsion prepared in Example 1 of this application;

[0021] Figure 3 Example 1 of this application was prepared to show the matrine release curves of the matrine sustained-release agent at different pH values;

[0022] Figure 4The following figures illustrate the deposition performance of the matrine sustained-release agent prepared in this application on Photinia leaves: Figure (A) shows the deposition effect of the matrine sustained-release agent, water, and matrine prepared in Example 1 on the surface of Photinia leaves; Figure (B) shows the deposition effect of the matrine sustained-release agent, water, and matrine prepared in Example 1 on the surface of Photinia leaves.

[0023] Figure (C) shows the deposition effect of the matrine sustained-release agent prepared in Examples 1-7 on the surface of Photinia leaves, and Figure (D) shows the deposition rate of the matrine sustained-release agent prepared in Examples 1-7 on the surface of Photinia leaves.

[0024] Figure 5 The contact angle diagrams of matrine sustained-release agent prepared in Example 1 of this application on the glass slide and the surface of photinia leaf are shown. Among them, Figure (A) is a physical diagram of the contact angle of different samples on the glass slide and the surface of photinia leaf, and Figure (B) is a diagram of the numerical values ​​of the contact angle of different samples on the glass slide and the surface of photinia leaf.

[0025] Figure 6 Figure 1 shows the antibacterial properties of the matrine sustained-release agent prepared in Example 1 of this application; wherein, Figure (A) is a comparison of the antibacterial activity of the prepared Pickering emulsion with the blank control and pure matrine solution, and Figure (B) is a comparison of the antibacterial rates of the prepared Pickering emulsion with the blank control and pure matrine solution. Detailed Implementation

[0026] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are implemented based on the technical solutions of the present invention, and it should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0027] In the following examples, matrine was purchased from Maclean's, with a purity of 98%; carboxymethyl cellulose (CMC) had a purity of 98%; sodium dihydrogen phosphate had a purity of 99%; disodium hydrogen phosphate had a purity of 99%; fatty amines were purchased from Aladdin; and hydrochloric acid was purchased from Sinopharm Chemical Reagent Co., Ltd., AR, with a purity of 99%.

[0028] Figure 1 The process flow diagram for preparing matrine sustained-release formulation includes the following steps:

[0029] (1) Dissolve matrine in deionized water and add HCl aqueous solution to adjust the pH;

[0030] (2) Carboxycellulose and fatty amine were added to the above solution in sequence and stirred and emulsified to obtain a dispersion;

[0031] (3) Add the dispersion to rubber seed oil and sonicate to obtain W / O / W Pickering emulsion.

[0032] Example 1

[0033] A method for preparing a matrine sustained-release agent includes the following steps:

[0034] (1) At room temperature, dissolve 15 mg of matrine in 30 mL of deionized water and add 0.1 M HCl aqueous solution to adjust the pH to 5.8.

[0035] (2) 10 mg of carboxycellulose (molecular weight 12000) and 15 mg of hexadecylamine were added to the above aqueous solution in sequence. After stirring evenly, the mixture was ultrasonically emulsified for 10 min (temperature controlled at 25℃) using a probe. After dialyzing the matrine molecules not included in the mixture using a dialysis bag, an amphiphilic gel dispersion loaded with matrine was obtained.

[0036] (3) Slowly add 30 mL of dispersion to 6 mL of rubber seed oil, and use an ultrasonic cell disruptor to sonicate the mixture again for 10 min (temperature controlled at 25℃) to obtain W / O / W Pickering emulsion.

[0037] Figure 2 The image shows the fluorescence inverted microscope characterization of the prepared matrine sustained-release emulsion. The emulsion droplets have smooth surfaces, are evenly distributed and do not show obvious aggregation, and are spherical or nearly spherical, which is consistent with the characterization of an emulsion. Therefore, it can be concluded that the Pickering emulsion has been successfully prepared.

[0038] Example 2

[0039] The difference from Example 1 is that the mass of matrine in step (1) is replaced with 30 mg, the mass of carboxycellulose in step (2) is replaced with 10 mg, and the mass of hexadecylamine is replaced with 20 mg, so as to obtain a W / O / W Pickering emulsion.

[0040] Example 3

[0041] The difference from Example 1 is that the hexadecylamine in step (2) is replaced with octadecylamine to obtain a W / O / W Pickering emulsion.

[0042] Example 4

[0043] The difference from Example 1 is that the molecular weight of carboxycellulose in step (2) is replaced with 15,000 to obtain a W / O / W Pickering emulsion.

[0044] Example 5

[0045] The difference from Example 1 is that the emulsification temperature in step (2) is changed to 28°C to obtain a W / O / W Pickering emulsion.

[0046] Example 6

[0047] The difference from Example 1 is that the volume of the dispersion in step (3) is replaced with 36 mL and the volume of the vegetable oil is replaced with 6 mL to obtain a W / O / W Pickering emulsion.

[0048] Example 7

[0049] The difference from Example 1 is that the secondary emulsification temperature in step (3) is changed to 28°C to obtain a W / O / W Pickering emulsion.

[0050] Example 8

[0051] The sustained-release behavior of the W / O / W Pickerling emulsion prepared in Example 1 was evaluated using dialysis. The method involved encapsulating 5 mL of the W / O / W Pickerling emulsion in a dialysis bag with a molecular weight cutoff of 8000, and then placing it in solutions prepared with sodium dihydrogen phosphate and disodium hydrogen phosphate to achieve three different pH values ​​(5.0, 6.8, and 7.4). Dialysis fluid was collected periodically (5 mL each time), and an equal volume of fresh medium was added. The actual concentration in the release solution was quantitatively detected using a UV spectrophotometer, and the cumulative drug release rate was calculated by time-concentration integration. Results are as follows: Figure 3 As shown.

[0052] Depend on Figure 3 It can be seen that in a neutral soil environment with a pH of 7.4, the emulsion retention rate remained above 80% after 12 hours, indicating that the emulsion has a good retention effect. In an acidic soil environment with a pH of 6.8, the retention rate was less than 30% after 12 hours, and the release rate was even faster, proving its acid-responsive release (i.e., rapid release under acidic conditions). In an acidic soil environment with a pH of 5.0, the retention rate was less than 30% after 12 hours, and the release rate was even faster, proving its acid-responsive release (i.e., rapid release under acidic conditions). Therefore, it has a good retention effect in a neutral soil environment, while exhibiting acid-responsive release under acidic conditions.

[0053] Example 9

[0054] Fresh photinia leaves were washed and air-dried. The initial weight of each leaf was recorded using a four-position balance, and then the leaves were immersed in different sample solutions from Examples 1–7 for 20 seconds. After immersion, the leaves were removed with tweezers, left to stand for 5 seconds, and weighed again. The deposition effect of the samples was evaluated based on the change in leaf weight before and after immersion. The results are as follows: Figure 4 As shown.

[0055] Depend on Figure 4(AB) is a comparative deposition performance diagram of the W / O / W Pickerling emulsion prepared in Example 1 and the solvents of each system on photinia leaves. The deposition rate of pure aqueous solution and pure matrine solution is only about 22%, while the deposition rate of Pickerling emulsion is about 34%. It can be seen that the prepared Pickerling emulsion has a better deposition effect than water and pure matrine solution.

[0056] Depend on Figure 4 (CD) is a comparative deposition rate diagram of the W / O / W Pickering emulsions prepared in Examples 1 to 7 on the leaf surface. From left to right, they are Examples 1 to 7, showing an increasing trend. Among them, the deposition rate of Example 7 is the highest, indicating that the W / O / W Pickering emulsion prepared in Example 7 has a better deposition rate effect.

[0057] Figure 5 The contact angle diagrams of the W / O / W Pickering emulsion prepared in Example 1 on the surfaces of a glass slide and a photinia leaf show that the prepared emulsion has good wetting effect on both the glass slide and the photinia leaf, with contact angles of less than 60°.

[0058] Example 10

[0059] Three parallel experiments were conducted using a blank group (without any additives), a pure matrine group, and a Pickering emulsion group from Example 1.

[0060] Experimental Preparation: Sterilize the laminar flow hood with ultraviolet light for 30 minutes beforehand to ensure a sterile experimental environment. Prepare the necessary culture medium (nutrient agar), petri dishes (90mm in diameter), inoculation loop, pipette, and the bacterial suspension to be tested (concentration adjusted to 10). 6 -10 7 Equipment such as CFU / mL, antibacterial substances, alcohol lamps, and constant temperature incubators were all sterilized by high-pressure steam (121℃, 20 minutes).

[0061] Culture medium preparation and sterilization: Accurately weigh each component according to the culture medium formula, add an appropriate amount of distilled water, heat and stir until completely dissolved, and adjust the pH to the suitable range (pH 7.0-7.2 for nutrient agar medium). Dispense the prepared culture medium into Erlenmeyer flasks and autoclave (121℃, 20 minutes). After sterilization, place the Erlenmeyer flasks in a 50-60℃ water bath for incubation.

[0062] Plate pouring procedure: In a laminar flow hood, take a sterilized petri dish, open one corner of the lid, and use a pipette to draw 15-20 mL of insulated culture medium. Quickly pour the medium into the dish and immediately replace the lid. Gently rotate the dish to evenly spread the culture medium across the entire bottom. Then, place the dish horizontally in the laminar flow hood and allow the culture medium to solidify (approximately 30-60 minutes).

[0063] Inoculation of bacterial suspension: For antibacterial experiments, the Oxford cup method or the spread plate method can be used. Taking the spread plate method as an example, use a pipette to draw 0.1 mL of the bacterial suspension to be tested and drop it onto the surface of the solidified culture medium. Use a sterile spreader to evenly spread the bacterial suspension over the entire surface of the culture medium and let it stand for 5-10 minutes to allow the bacterial suspension to be completely absorbed. If using antibacterial substances, after spreading the bacterial suspension, filter paper discs (6 mm in diameter, sterilized) soaked in the antibacterial substance can be attached to the surface of the culture medium. 3-5 discs can be placed on each petri dish, evenly distributed.

[0064] Cultivation and Observation: Invert the inoculated petri dishes (to prevent condensation from contaminating the colonies) and place them in a constant temperature incubator at 37°C. Remove the petri dishes after 1, 2, 3, 4, 5, 6, and 7 days of cultivation, and observe and record the colony growth. During observation, use a colony counter or a ruler to measure the diameter of each colony (in mm). For contiguous colonies, record their spread.

[0065] Antibacterial inhibition rate calculation: The antibacterial inhibition rate is calculated based on the colony diameter measured at different time points. The formula for calculating the antibacterial inhibition rate is: Antibacterial inhibition rate (%) = (Average colony diameter of control group - Average colony diameter of experimental group) / Average colony diameter of control group × 100%. Wherein, the control group consists of petri dishes without added antibacterial substances, and the experimental group consists of petri dishes with added antibacterial substances. If no antibacterial substances are used, and only changes in colony size at different time points are observed to determine growth, the colony diameter data at each time point can be directly recorded.

[0066] The test results are from Figure 6 The results showed that the antibacterial rate of Pickering emulsion remained at around 25%, while that of pure matrine solution was slightly lower, fluctuating at around 22%, and that of pure aqueous solution was between 14-18%. This demonstrates that Pickering emulsion still has a good antibacterial effect despite having a high deposition rate.

[0067] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a matrine sustained-release agent, characterized in that: First, matrine is dissolved in an acidic aqueous solution. Then, carboxycellulose and fatty amine are added to the solution in sequence and stirred and emulsified to obtain a dispersion. Finally, the dispersion is added to vegetable oil for secondary emulsification to obtain a W / O / W Pickering emulsion.

2. The method for preparing matrine sustained-release agent according to claim 1, characterized in that: The pH of the acidic solution is 5.5 to 7.

4.

3. The method for preparing matrine sustained-release agent according to claim 1, characterized in that: The mass ratio of matrine, carboxycellulose, and fatty amine is 3:1:2 to 3:2:

3.

4. The method for preparing matrine sustained-release agent according to claim 1, characterized in that: The fatty amine has 12 to 18 carbon atoms, with an amino group at position 1.

5. The method for preparing matrine sustained-release agent according to claim 1, characterized in that: The molecular weight of the carboxycellulose is 12,000 to 15,000.

6. The method for preparing matrine sustained-release agent according to claim 1, characterized in that: The emulsification temperature is 25–30°C, and the time is 10 minutes.

7. The method for preparing matrine sustained-release agent according to claim 1, characterized in that: The volume ratio of the dispersion to the vegetable oil is 5-6:

1.

8. The method for preparing matrine sustained-release agent according to claim 1, characterized in that: The secondary emulsification is performed at a temperature of 25–30°C for 10 minutes.

9. The matrine sustained-release agent prepared according to any one of claims 1 to 7 is obtained.

10. The application of the matrine sustained-release agent according to claim 8 in pesticide adjuvants.

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