Preparation method of 2,4-di-tert-butyl phenol nanoemulsion and application thereof

Abstract

The application relates to the fields of agricultural biotechnology and pesticide preparation technology, and discloses a preparation method and application of 2,4-di-tert-butyl phenol nanoemulsion, which comprises the following steps: 2,4-di-tert-butyl phenol is dissolved in ethyl acetate to form an oil phase mixture; the obtained oil phase mixture is mixed with Tween 80 under magnetic stirring, stirring is performed until the mixture is fully mixed, an oil phase-emulsifier system is obtained after internal gas is removed through ultrasonic treatment, then the oil phase-emulsifier system is slowly added into 8 times the volume of deionized water under magnetic stirring at 45 DEG C, and mutual dissolution of the water phase and the oil phase is promoted through ultrasonic treatment for 40 min to obtain a pre-emulsion. The obtained pre-emulsion is emulsified through a high-pressure homogenizer, 20000 psi, and circulation is performed for 8 times to obtain the nanoemulsion. The nanoemulsion prepared by the application has a nanoscale particle size, the system presents good kinetic stability, is not prone to delamination, precipitation or Ostwald ripening, and shows high storage stability.

Description

Technical Field

[0001] This invention relates to the fields of agricultural biotechnology and pesticide formulation technology, and in particular to the preparation method and application of 2,4-di-tert-butylphenol nanoemulsion. Background Technology

[0002] Soybeans are susceptible to various fungal diseases during cultivation, leading to significant yield reductions. Major diseases include anthracnose, rust, wilt, and damping-off caused by fungi such as *Anthracnose fungi*, *Potamogeton crispus*, *Fusarium*, and *Rhizoctonia solani*. Currently, the control of soybean fungal diseases mainly relies on chemical fungicides. However, traditional synthetic fungicides pose risks of environmental pollution, residues, and induction of drug-resistant bacteria, thus limiting their use. Therefore, there is an urgent need to develop highly effective biological fungicides to balance the relationship between antimicrobial yield enhancement and biosafety.

[0003] 2,4-Di-tert-butylphenol is a small-molecule phenolic compound widely found in plants and has been shown to have good antibacterial activity in recent years. However, its poor water solubility and chemical instability limit its application in practical agricultural production.

[0004] Nanoemulsions, as a novel drug delivery system, possess advantages such as small particle size, high stability, good solubilization effect, and improved bioavailability. Preparing 2,4-di-tert-butylphenol into a nanoemulsion holds promise for overcoming its existing shortcomings and improving its efficacy while reducing dosage. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing a method for preparing 2,4-di-tert-butylphenol nanoemulsions and their applications.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The preparation method of 2,4-di-tert-butylphenol nanoemulsion includes the following steps:

[0008] S1. Dissolve 2,4-di-tert-butylphenol in ethyl acetate to form an oil phase mixture with a concentration of 0.8-1.4 g / mL;

[0009] S2. Under magnetic stirring at 40-60℃, the oil phase mixture obtained in step S1 is mixed with Tween 80 and stirred until fully mixed. After removing the internal gas by ultrasound, an oil phase-emulsifier system is obtained.

[0010] S3. Under stirring at 40-50℃, add the oil phase-emulsifier system obtained in step S2 to deionized water, stir until fully mixed, and then sonicate to obtain a pre-emulsion. The amount of oil phase-emulsifier system added is 8%-20% of the volume of the pre-emulsion.

[0011] S4. The pre-emulsion is emulsified by a high-pressure homogenizer and cycled 5-10 times at 20,000 psi to obtain the 2,4-di-tert-butylphenol nanoemulsion.

[0012] Preferably, the 2,4-di-tert-butylphenol nanoemulsion obtained in step S4 comprises: 1%-10% by mass of 2,4-di-tert-butylphenol, 7%-10% by volume of ethyl acetate, 1%-5% by volume of Tween 80, and the balance being deionized water.

[0013] Preferably, in step S1, the concentration of 2,4-di-tert-butylphenol in the oil phase mixture is 1.0 g / mL.

[0014] Preferably, the stirring time in steps S2 and S3 is 3-6 h.

[0015] Preferably, in step S2, the stirring time is 4 hours under magnetic stirring at 40°C.

[0016] Preferably, in step S3, the stirring time is 4 hours at 45°C.

[0017] Preferably, in step S3, the duration of ultrasonic treatment is 30-60 min.

[0018] Preferably, in step S3, the duration of ultrasonic treatment is 40 min.

[0019] Preferably, in step S4, the number of cycles is 8.

[0020] A 2,4-di-tert-butylphenol nanoemulsion was prepared using the method described above.

[0021] The application of the above-mentioned 2,4-di-tert-butylphenol nanoemulsion in the preparation of agents for treating soybean fungal diseases.

[0022] Preferably, the 2,4-di-tert-butylphenol nanoemulsion is diluted with water 500 to 1000 times and then applied to soybeans by foliar spraying.

[0023] Preferably, the soybean fungal disease is soybean anthracnose.

[0024] The beneficial effects of this invention are as follows:

[0025] 1. High dispersibility and stability. The nanoemulsion prepared by this invention has nanoscale particle size, exhibits good kinetic stability, is not prone to stratification, precipitation or Ostwald ripening, and demonstrates high storage stability.

[0026] 2. Improved bioavailability of 2,4-di-tert-butylphenol. Since 2,4-di-tert-butylphenol itself has poor water solubility, preparing it as a nanoemulsion can improve its solubility and promote its adhesion, penetration, and translocation on plant leaf surfaces, thereby enhancing its effectiveness in controlling fungal diseases.

[0027] 3. Environmentally friendly and improves pesticide safety. Using water as the continuous phase reduces the use of organic solvents and the amount of active ingredient required, thus helping to reduce pesticide residues and environmental pollution.

[0028] 4. It possesses antibacterial activity and can be used to treat soybean fungal diseases. The nanoemulsion prepared in this invention has the ability to inhibit the growth of pathogenic fungi, effectively inhibiting the growth of pathogens such as *Anthracnose spp.*, and has a therapeutic effect on soybean anthracnose. Attached Figure Description

[0029] Figure 1 The figure shows the characterization results of the 2,4-di-tert-butylphenol nanoemulsion prepared in Example 1 of this invention; where A is a scanning electron microscope image of the nanoemulsion; B is the particle size distribution of the nanoemulsion; and C is the zeta potential test result of the nanoemulsion.

[0030] Figure 2 The results show the stability test results of the nanoemulsion, where A represents the state of the nanoemulsion after different ultrasonic treatment times; and B represents the state of the nanoemulsion after different centrifugation times.

[0031] Figure 3 The results of the in vitro antibacterial test of the nanoemulsion are shown in Figure 1. In Figure 2, A represents the colony growth status under different treatment conditions, and B represents the corresponding statistical results of the antibacterial rate.

[0032] Figure 4 The results of the test on the therapeutic effect of nanoemulsion on soybean anthracnose are shown. In this paper, A represents the state of soybean leaves under different treatment conditions, and B represents the statistical results of the incidence rate. Detailed Implementation

[0033] To provide a clearer understanding of the technical features, objectives, and beneficial effects of this invention, the technical solution of this invention is described in detail below, but this should not be construed as limiting the scope of implementation of this invention. Unless otherwise specified, the methods used in this invention are conventional methods in this technical field. In this invention, materials, reagents, or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0034] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0035] Example 1: Step 1: Weigh 50 g of 2,4-di-tert-butylphenol and dissolve it in ethyl acetate to prepare an oil phase mixture with a concentration of 1.0 g / mL.

[0036] Step 2: Measure 5 mL of Tween 80 and add it to the oil phase. Stir magnetically at 60℃ and 1000 rpm for 4 h. Then, use a KQ-250DE ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.) to sonicate at 60℃ and 30% intensity for 40 min to make the system clear and transparent, thus obtaining the emulsifier-oil phase mixture system.

[0037] Step 3: Slowly add 55 mL of the emulsifier-oil phase mixture to 445 mL of deionized water, and continuously stir magnetically at 45°C and 1000 rpm until the mixture is homogeneous. Then, sonicate at 45°C and 30% intensity for 40 min to obtain the pre-emulsion.

[0038] Step 4: Homogenize the pre-emulsion using a high-pressure homogenizer (Phd D-3L VELP) at 20,000 psi for 8 cycles to obtain a milky white 2,4-di-tert-butylphenol nanoemulsion.

[0039] Example 2: Step 1: Weigh 40 g of 2,4-di-tert-butylphenol and dissolve it in ethyl acetate to prepare an oil phase mixture with a concentration of 0.8 g / mL.

[0040] Step 2: Measure 5 mL of Tween 80 and add it to the oil phase. Stir magnetically for 3 h at 40℃ and 1000 rpm. Then, use a KQ-250DE ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.) to sonicate at 40℃ and 30% intensity for 30 min to make the system clear and transparent, thus obtaining the emulsifier-oil phase mixture system.

[0041] Step 3: Slowly add 55 mL of the emulsifier-oil phase mixture to 445 mL of deionized water, and continuously stir magnetically for 3 h at 40℃ and 1000 rpm until the mixture is homogeneous. Then, sonicate at 40℃ and 30% intensity for 30 min to obtain the pre-emulsion.

[0042] Step 4: Homogenize the pre-emulsion using a high-pressure homogenizer (Phd D-3L VELP) at 20,000 psi for 5 cycles to obtain a milky white 2,4-di-tert-butylphenol nanoemulsion.

[0043] Example 3: Step 1: Weigh 50 g of 2,4-di-tert-butylphenol and dissolve it in ethyl acetate to prepare an oil phase mixture with a concentration of 1.4 g / mL.

[0044] Step 2: Measure 5 mL of Tween 80 and add it to the oil phase. Stir magnetically for 6 h at 60℃ and 1000 rpm. Then, use a KQ-250DE ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.) to sonicate at 60℃ and 30% intensity for 60 min to make the system clear and transparent, thus obtaining the emulsifier-oil phase mixture system.

[0045] Step 3: Slowly add 40 mL of the emulsifier-oil phase mixture to 460 mL of deionized water, and continuously stir magnetically at 50°C and 1000 rpm until the mixture is homogeneous. Then, sonicate at 50°C and 30% intensity for 60 min to obtain the pre-emulsion.

[0046] Step 4: Homogenize the pre-emulsion using a high-pressure homogenizer (Phd D-3L VELP) at 20,000 psi for 10 cycles to obtain a milky white 2,4-di-tert-butylphenol nanoemulsion.

[0047] Test Example 1: This test example characterizes the 2,4-di-tert-butylphenol nanoemulsion prepared in Example 1. The results are shown in [link to test example]. Figure 1 As shown.

[0048] Take 20 mL of the 2,4-di-tert-butylphenol nanoemulsion prepared in Example 1 and place it in a sample vial. Dilute it to 10 mL with deionized water using the multiple dilution method. 1 10 2 10 3 10 4 The samples were diluted to a concentration of 10 times and placed in sample vials to observe their water solubility and flowability at different dilution ratios. Then, a 10-fold dilution was selected. 1 For samples diluted 10 times, the particle diameter of the nanoemulsion was measured using a Zeta potential and particle size analyzer (Malvern), and the average particle size was analyzed and recorded; a 10-fold dilution was selected. 3 The stability of the system was evaluated by performing zeta potential tests on samples of multiple times the original volume.

[0049] Finally, 2 μL of nanoemulsion was dropped onto the surface of the silicon wafer, allowed to air dry naturally, and then sputtered with gold. The morphology of the nanoemulsion was observed using a field emission scanning electron microscope (Regulus 8100).

[0050] Test Example 2: This test example examines the ultrasonic and centrifugal stability of the 2,4-di-tert-butylphenol nanoemulsion prepared in Example 1. The results are shown in [link to test example]. Figure 2 ;

[0051] Take 1 mL of nanoemulsion into centrifuge tubes and treat them under different ultrasonic time conditions. The ultrasonic time is set to 0, 1, 2, 3, 4, 5 and 10 min. After treatment, observe and record the state of the sample to evaluate the stability of the nanoemulsion under ultrasonic disturbance.

[0052] The nanoemulsion samples were centrifuged at 12000 rpm for 0, 1, 2, 3, 4, 5, and 10 min, respectively. The state of the nanoemulsion after centrifugation was observed and recorded to evaluate the centrifugation stability of the system.

[0053] Test results showed that no significant precipitation or turbidity occurred in the nanoemulsion when the ultrasonic treatment time was extended to 10 min. During centrifugation, no obvious stratification was observed within 4 min, while slight stratification occurred when the centrifugation time exceeded 4 min. The relevant results are as follows: Figure 2 As shown.

[0054] Test Example 3: This test example evaluates the in vitro antibacterial activity of the 2,4-di-tert-butylphenol nanoemulsion prepared in Example 1. The relevant results are shown in [link to test example]. Figure 3 .

[0055] Weigh 4.6 g of potato dextrose agar (PDA) and add it to 100 mL of distilled water to prepare PDA medium. Sterilize the medium by steam at 121°C for 20 min. When the medium temperature drops to approximately 40°C, add 20–200 μL of nanoemulsion to each medium, stir thoroughly, and then pour the medium into petri dishes to solidify naturally, thus preparing test plates of different concentrations. Plates prepared by adding an equal volume of solvent nanoemulsion to the medium serve as controls.

[0056] Subsequently, a 5 mm diameter mycelial cake was inoculated in the center of each plate and incubated at 23°C for 10 days. The mycelial cake used was *Anthracnose spp.* Colletotrichum truncatum After cultivation, the colony diameter was measured, and the antibacterial rate of the nanoemulsion was calculated. The antibacterial rate was calculated using the following formula:

[0057]

[0058] in, The diameter of colonies on the control plate. To test the diameter of bacterial colonies on plates, the concentration of nanoemulsions that completely inhibited the growth of *Anthrax flavomarginata* was used as the minimum inhibitory concentration (MIC). Each experiment was repeated three times, and results are expressed as mean ± standard deviation.

[0059] Test Example 4: This test example was used to evaluate the therapeutic effect of the 2,4-di-tert-butylphenol nanoemulsion prepared in Example 1 on soybean anthracnose. The relevant results are shown in... Figure 4 .

[0060] Anthracnose bacterium was inoculated onto PDA medium and cultured at 25°C for 10 days to allow the bacteria to fully colonize the plates. Subsequently, peat moss and vermiculite were mixed evenly at a volume ratio of 1:2, sterilized by high-temperature steam at 121°C for 20 minutes, cooled, and then dispensed into small flower pots for later use.

[0061] Select soybean seeds with intact seed coats and uniform size, and sow them in flowerpots at a depth of about 2 cm. After watering, place them in an artificial climate chamber for cultivation until the first pair of true leaves of the soybeans are fully unfolded. The cultivation period is about 10 days. The cultivation conditions are: light temperature 25℃, dark temperature 22℃, and light-dark cycle of 16 h:8 h.

[0062] A 5 mm diameter mycelium was inoculated onto soybean leaves, and 2 mL of nanoemulsions at concentrations of 0.5 MIC, 1 MIC, and 2 MIC, respectively, along with an equal volume of control solution, were sprayed. The mycelium used was *Anthracnose spp.* Colletotrichum truncatum After culturing for another 10 days, photograph and record the leaf lesions, and measure the lesion area and leaf area. Calculate the lesion ratio using the following formula:

[0063]

[0064] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. 2,4-Di-tert-butylphenol nanoemulsion in the preparation of agents for controlling soybean fungal diseases, wherein the soybean fungal disease is soybean anthracnose caused by *Anthracnose spp.*, and the preparation method of the 2,4-di-tert-butylphenol nanoemulsion includes the following steps: S1. Dissolve 2,4-di-tert-butylphenol in ethyl acetate to form an oil phase mixture with a concentration of 0.8-1.4 g / mL; S2. Under magnetic stirring at 40-60℃, the oil phase mixture obtained in step S1 is mixed with Tween 80 and stirred until fully mixed. After removing the internal gas by ultrasound, an oil phase-emulsifier system is obtained. S3. Under stirring at 40-50℃, add the oil phase-emulsifier system obtained in step S2 to deionized water, stir until fully mixed, and then sonicate to obtain a pre-emulsion. The amount of oil phase-emulsifier system added is 8%-20% of the volume of the pre-emulsion. S4. The pre-emulsion is emulsified by a high-pressure homogenizer and cycled 5-10 times at 20,000 psi to obtain the 2,4-di-tert-butylphenol nanoemulsion.

2. Use according to claim 1, characterized in that, In step S1, the concentration of 2,4-di-tert-butylphenol in the oil phase mixture is 1.0 g / mL.

3. Use according to claim 1, characterized in that, The 2,4-di-tert-butylphenol nanoemulsion obtained in step S4 comprises: 1%-10% by mass of 2,4-di-tert-butylphenol, 7%-10% by volume of ethyl acetate, 1%-5% by volume of Tween 80, and the balance being deionized water.

4. Use according to claim 1, characterized in that, In step S2, the stirring time is 4 hours under magnetic stirring at 40°C.

5. The use according to claim 1, characterized in that, In step S3, the stirring time is 4 hours at 45°C.

6. Use according to claim 1, characterized in that, In step S3, the duration of ultrasonic treatment is 30-60 minutes.

7. Use according to claim 1, characterized in that, The 2,4-di-tert-butylphenol nanoemulsion was diluted 500 to 1000 times with water and then applied to soybeans by foliar spraying.

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