Carvacrol-loaded Pickering emulsion as well as preparation method and application thereof

Abstract

The invention discloses a carvacrol-loaded Pickering emulsion as well as a preparation method and application thereof, and belongs to the technical field of insecticides. The preparation method of the carvacrol-loaded Pickering emulsion comprises the following steps: firstly, grafting cholesterol onto sodium alginate to obtain cholesterol grafted sodium alginate; then, jointly dispersing the cholesterol grafted sodium alginate and SiO2 nanoparticles in water, so that the cholesterol grafted sodium alginate is adsorbed on the SiO2 nanoparticles, and modified SiO2 nanoparticles are obtained; and adding the modified SiO2 nanoparticles and carvacrol into an oil phase, and preparing the Pickering emulsion by matching with a water phase, so as to obtain the carvacrol-loaded Pickering emulsion. According to the invention, the Pickering emulsion technology is utilized, the stabilizer is combined to modify the SiO2 nanoparticles, and the volatile carvacrol is coated in the emulsion, so that the stability of the carvacrol is improved, and the effectiveness of the carvacrol is also improved.

Description

Technical Field

[0001] This invention belongs to the field of insecticide technology, specifically relating to a Pickering emulsion loaded with carvacrol, its preparation method, and its application. Background Technology

[0002] Traditional pesticide formulations, especially emulsifiable concentrates and some suspension concentrates, heavily rely on large amounts of organic solvents and surfactants. This not only increases production costs but also leads to a series of serious problems, including environmental pollution, excessive residues in agricultural products, and non-target biotoxicity. Developing highly efficient, low-toxicity, and environmentally friendly new pesticide formulations has become an important direction for sustainable agricultural development. Pickering emulsion technology, which uses solid particles as stabilizers, shows great potential in this regard. Unlike traditional emulsions that rely on amphiphilic molecules (surfactants) to reduce interfacial tension, Pickering emulsions utilize nano- or micro-sized solid particles (such as silica, clay minerals, polymers, and carbon materials) that are irreversibly adsorbed at the oil-water interface, forming a dense physical barrier that greatly stabilizes the emulsion droplets. This unique stabilization mechanism endows Pickering emulsions with a series of superior properties: extremely high stability (anti-agglomeration, anti-Ostwald ripening), extremely low interfacial toxicity (requiring no or only a very small amount of traditional surfactants), tunable interfacial properties, and environmentally friendly performance. Pickering emulsion technology provides an innovative platform for building a new generation of intelligent and precise pesticide delivery systems. Summary of the Invention

[0003] The purpose of this invention is to provide a carvacrol-loaded Pickering emulsion, its preparation method, and its application. Cholesterol is first grafted onto sodium alginate, and then the grafted product is used to modify silica nanoparticles to obtain modified silica nanoparticles. These modified silica nanoparticles are then used as a stabilizer in conjunction with carvacrol to prepare a Pickering emulsion, which is the carvacrol-loaded Pickering emulsion.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] One of the technical solutions of this invention is to provide a method for preparing a Pickering emulsion loaded with carvacrol, comprising the following steps:

[0006] First, cholesterol is grafted onto sodium alginate to obtain cholesterol-grafted sodium alginate. Then, the cholesterol-grafted sodium alginate and SiO2 nanoparticles are co-dispersed in water, allowing the cholesterol-grafted sodium alginate to adsorb onto the SiO2 nanoparticles, resulting in modified SiO2 nanoparticles. The modified SiO2 nanoparticles and carvacrol are added to the oil phase and combined with the aqueous phase to prepare a Pickering emulsion, which is the carvacrol-loaded Pickering emulsion.

[0007] Preferably, the preparation steps of the cholesterol-grafted sodium alginate include: dissolving sodium alginate and anhydrous p-toluenesulfonic acid together in 38 mL of formic acid / N,N-dimethylformamide mixed solvent, and stirring at 55 °C for 30 min to protonate; then adding 4-dimethylaminopyridine (DMAP), N,N-dicyclohexylcarbodiimide (DCC) and cholesterol dissolved in chloroform to the system, and reacting at 40 °C for 24 h to obtain cholesterol-grafted sodium alginate.

[0008] More preferably, the ratio of sodium alginate, anhydrous p-toluenesulfonic acid, formic acid / N,N-dimethylformamide mixed solvent, 4-dimethylaminopyridine, N,N-dicyclohexylcarbodiimide, and cholesterol is 1.0g:0.324g:38mL:0.475g:0.4g:1.0g; and the volume ratio of formic acid to N,N-dimethylformamide in the formic acid / N,N-dimethylformamide mixed solvent is 10:9.

[0009] Preferably, the mass ratio of cholesterol-grafted sodium alginate to SiO2 nanoparticles is 1:5.

[0010] Preferably, the adsorption of cholesterol-grafted sodium alginate onto SiO2 nanoparticles further includes a treatment step to reduce the particle size of the modified SiO2 nanoparticles.

[0011] Preferably, the carvacrol is carvacrol stock solution or carvacrol-containing emulsion.

[0012] Preferably, the concentration of modified SiO2 nanoparticles in the oil phase is 10 mg / mL.

[0013] The second technical solution of the present invention provides a Pickering emulsion loaded with carvacrol prepared according to the above-mentioned method for preparing Pickering emulsion loaded with carvacrol.

[0014] The third technical solution of the present invention provides the application of the above-mentioned Pickering emulsion loaded with carvacrol in the preparation of an agent for controlling citrus psyllids.

[0015] The beneficial technical effects of the present invention are as follows:

[0016] This invention utilizes Pickering emulsion technology to encapsulate volatile carvacrol in an emulsion, thereby improving the stability of carvacrol. In addition, by using cholesterol-grafted sodium alginate-modified SiO2 nanoparticles as a stabilizer, the prepared Pickering emulsion can penetrate more easily into plant leaves, which can improve the effectiveness of carvacrol encapsulation. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 Sodium alginate and CSAD 1 H-NMR spectrum.

[0019] Figure 2 The FT-IR spectra of sodium alginate and CSAD are shown.

[0020] Figure 3 The image shows the modified SiO2 nanoparticles and their particle size distribution.

[0021] Figure 4 The image shows the Zate potential distribution of the modified SiO2 nanoparticles and the SiO2 nanoparticles.

[0022] Figure 5 The Zate potential distribution diagrams for the picking emulsions numbered one to six in Table 1 of Example 3 are shown.

[0023] Figure 6 The PDI values ​​are for the picking emulsions numbered one to six in Table 1 of Example 3.

[0024] Figure 7 The particle size distribution diagram of the picking emulsions No. 1 to No. 6 in Table 1 of Example 3 is shown.

[0025] Figure 8 Photos taken on days 1 to 7, showing the No. 1 carvacrol stock solution emulsion and the No. 4 5% carvacrol sample emulsion stored in the refrigerator for 7 days.

[0026] Figure 9 The relationship between the cumulative release rate of carvacrol in the No. 1 carvacrol stock solution emulsion and time.

[0027] Figure 10 The relationship between the cumulative release rate of carvacrol in the emulsion of 5% carvacrol sample No. 4 and time.

[0028] Figure 11 These are photographs of the morphology and growth status of citrus leaves in the control group from day 1 to day 14 during the test of the effect of the sample emulsion on the growth of citrus leaves.

[0029] Figure 12 These are photos of the morphology and growth status of citrus leaves 1–14 days after spraying the No. 1 carvacrol stock solution emulsion in a test on the effect of the sample emulsion on the growth of citrus leaves.

[0030] Figure 13 These are photos of the morphology and growth status of citrus leaves 1–14 days after spraying the No. 4 5% carvacrol sample emulsion, in a test on the effect of the sample emulsion on the growth of citrus leaves.

[0031] Figure 14 The contact angle of the No. 1 carvacrol stock solution emulsion dropped onto citrus leaves.

[0032] Figure 15 The contact angle of sample emulsion No. 4 (5% carvacrol) dropped onto citrus leaves.

[0033] Figure 16 The results of the spreading properties test of different samples on citrus leaves.

[0034] Figure 17 The results show the adhesion of carvacrol stock solution emulsion No. 1 and carvacrol sample emulsion No. 4 to citrus leaves.

[0035] Figure 18 The fluorescence values ​​of the sample emulsion of Cy5-labeled nanomaterials and the citrus leaves and adjacent leaves after spraying with free Cy5 were measured.

[0036] Figure 19 Fluorescence values ​​of citrus stems sprayed with Cy5-labeled 5% carvacrol sample emulsion and free Cy5.

[0037] Figure 20 The mortality rate of citrus psyllids in different treatment groups during a bioactivity test for citrus psyllids.

[0038] Figure 21 The distribution of citrus psyllids on citrus seedlings after 48 hours in different treatment groups during the bioactivity test of citrus psyllids. Detailed Implementation

[0039] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0040] It should be noted that any aspects not described in detail in this invention are conventional practices in the field and are not the focus of this invention.

[0041] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0042] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0043] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0044] Unless otherwise specified, room temperature in this invention refers to a temperature of 20±10℃.

[0045] Example 1

[0046] Synthesis of cholesterol-grafted sodium alginate (CSAD):

[0047] Sodium alginate (M) w =169920, 1.0 g) and anhydrous p-toluenesulfonic acid (0.324 g) were dissolved together in 38 mL of a formic acid / N,N-dimethylformamide (volume ratio 10:9) mixed solvent, and partially protonated by stirring at 55 °C for 30 min. Subsequently, DMAP (0.475 g), DCC (0.4 g), and 2 mL of cholesterol solution dissolved in chloroform (cholesterol content 0.5 g / mL) were added to the system, and the reaction was carried out at 40 °C for 24 h to allow the carboxylic acid group (–COOH) of protonated sodium alginate to undergo an esterification reaction with the hydroxyl group of cholesterol. After the reaction was completed, ethanol was added to the mixture to precipitate the product, which was then separated by centrifugation, washed thoroughly with anhydrous ethanol, and neutralized by adding a 1.5% sodium carbonate and sodium bicarbonate mixed solution. Finally, the product solution was dialyzed against distilled water for 3 days, and then freeze-dried to obtain purified CSAD. The CSAD product appears as a pale yellow, semi-transparent film that is difficult to grind. It is soluble in water but insoluble in ethanol, methanol, etc. The aqueous solution has a high viscosity, and the yield is approximately 20%.

[0048] The structure of the prepared CSAD was determined using the following method:

[0049] Nuclear magnetic resonance analysis (NMR) 1H-NMR: Sodium alginate and CSAD were dissolved in heavy water (D2O) and measured separately. 1 H-NMR spectra were used to determine the polymer molecular structure and degree of substitution. All samples were at a concentration of 5 mg / mL, and measurements were performed at room temperature using water peak processing. 1 The degree of substitution of the deoxycholic acid group is calculated by integrating H-NMR.

[0050] Infrared spectroscopy analysis (FT-IR): To provide a comparative explanation, dried sodium alginate samples were mixed and ground with KBr, then compressed into tablets. Fourier transform infrared spectroscopy was used to characterize sodium alginate and CSAD separately to determine the functional group structures and binding modes of the components. The number of scans was 32, and the resolution was 4 cm⁻¹. -1 .

[0051] Figure 1 Sodium alginate and CSAD 1 H-NMR spectrum (the top one is sodium alginate) 1 H-NMR spectrum, below is CSAD 1 H-NMR spectrum); Figure 2 FT-IR spectra of sodium alginate (Alg) and CSAD.

[0052] Figures 1-2 The display shows 1611cm. -1 The absorption peak at 2928 cm⁻¹ is the asymmetric stretching vibration peak of -COO-. -1 The vibrational absorption peak at 3445 cm⁻¹ is the stretching vibration peak of the methylene-CH group on the sodium alginate backbone. -1 The absorption peak at 1737 cm⁻¹ is due to the stretching vibration of -OH. The CSAD peak at 1737 cm⁻¹ is a new addition to the spectrum relative to Alg. -1 The absorption peak is the stretching vibration peak of the ester group, and it is also at 2851 cm⁻¹. -1 The absorption peak observed is due to the C-H stretching vibration of the methylene group. Since sodium alginate itself does not contain a methylene group, this indicates that the carboxyl group of sodium alginate has undergone esterification with the hydroxyl group of cholesterol. The proton peaks from 3.2 ppm to 5.5 ppm are caused by the H atoms in the native sodium alginate chain. Compared to the spectrum of sodium alginate, some additional proton peaks were observed in the range of 1.0 ppm to 2.0 ppm. These proton peaks are attributed to the methylene protons of the cholesteryl group, indicating that CSAD has been successfully synthesized.

[0053] Example 2

[0054] Preparation of modified SiO2 nanoparticles (CSAD-SiO2):

[0055] The raw materials were mixed according to the following dosage: 20 mg CSAD, 100 mg SiO2 nanoparticles, and 20 mL distilled water. The mixture was stirred at 1000 rpm for 18 min, and then sonicated in a water bath at room temperature for 30 min at a power of 120 W. Surface modification of the SiO2 nanoparticles was achieved through electrostatic adsorption. Pre-treatment was then performed by shearing for 4 min using a shear emulsifier in D mode to initially reduce the particle size of the modified SiO2 nanoparticles. A certain amount of the modified SiO2 nanoparticle dispersion was taken, centrifuged at 12000 rpm for 15 min, and the precipitate was washed three times with ultrapure water. After pre-freezing at -80℃ for 12 h, the precipitate was then freeze-dried using a vacuum freeze dryer to obtain lyophilized modified SiO2 nanoparticle powder.

[0056] The prepared modified SiO2 nanoparticles were characterized using the following methods:

[0057] The particle size and Zate potential of modified SiO2 nanoparticles and SiO2 nanoparticle raw materials were determined by dynamic light scattering at 25℃. Based on the experimental results, particle size distribution and Zate potential distribution of SiO2 were plotted.

[0058] Figure 3 The image shows the modified SiO2 nanoparticles and their particle size distribution. Figure 4 The image shows the Zate potential distribution of the modified SiO2 nanoparticles and the SiO2 nanoparticles.

[0059] Figures 3-4 The results show that the zeta potentials of both CSAD-SiO2 and SiO2 nanoparticles are below -20 mV, indicating an initial unstable state. However, they become relatively stable when the zeta particle size of CSAD-SiO2 is smaller than that of SiO2 nanoparticles. Mixing CSAD and SiO2 nanoparticles leads to physical adsorption, resulting in modified SiO2 nanoparticles, i.e., CSAD-SiO2. The fact that the CSAD-SiO2 particle size is smaller than that of SiO2 nanoparticles demonstrates successful adsorption.

[0060] Example 3

[0061] Preparation of Pickering emulsion loaded with carvacrol:

[0062] 100 mg of modified SiO2 nanoparticles were dissolved in 10 mL of vegetable oil, and this, along with carvacrol stock solution or a commercially available 5 wt% carvacrol sample emulsion (Shanxi Dewei Herbal Biotechnology Co., Ltd.), was used as the oil phase. Subsequently, 0.125 mL of sterile deionized water was added to the oil phase with stirring. A water-in-oil (W / O) primary emulsion was prepared by ultrasonic treatment (Branson Digital Sonifier, US, total time = 120 s, power = 45%, interval time = 4 s). This primary emulsion was then added to 8 mL of an external aqueous phase containing PVA (PVA concentration of 2 g / 100 mL), and ultrasonic treatment was performed using the same procedure to obtain the final W / O / W multiple emulsion. The multiple emulsion was then added to 4 mL of a PVA-containing solution (PVA concentration of 2 g / 100 mL) and stirred at room temperature for 4 h to obtain a stable reverse-phase carvacrol-loaded pickering emulsion.

[0063] The oil phase ratio of the stable reverse-phase loaded carvacrol-containing pickering emulsion in Example 3 is shown in Table 1.

[0064] Table 1. Oil phase ratio of stable reverse-phase loaded carvacrol pickering emulsion

[0065]

[0066] The six pickering emulsions prepared in Example 3 were characterized as follows:

[0067] The particle size and Zate potential of the prepared pickering emulsion were determined by dynamic light scattering at 25℃. Based on the experimental results, particle size distribution and Zate potential distribution of the pickering emulsion were plotted.

[0068] Figure 5 Zate potential distribution of pickering emulsions numbered one to six (from left to right, numbered one to six). Figure 6 The PDI values ​​are for pickering lotions numbered one to six (from left to right, numbered one to six). Figure 7 This is a particle size distribution diagram of pickering emulsions numbered one through six.

[0069] pass Figures 5-7 Based on the experimental results, pickering emulsions No. 1 and No. 4 were selected as the subjects of investigation (the particle size distribution of the two is close to a normal distribution).

[0070] The stability of the selected carvacrol stock solution emulsion (No. 1) and the 5% carvacrol sample emulsion (No. 4) was observed. Both were stored in a refrigerator at 4°C for 7 days, and photos were taken and recorded daily. The results are shown below. Figure 8(Photos from left to right, showing days 1 to 7). From Figure 8 As can be seen, the No. 4 5% carvacrol sample emulsion is more stable than the No. 1 carvacrol stock emulsion, and no stratification phenomenon was observed after being placed in the refrigerator for 7 days.

[0071] Further testing was conducted on the carvacrol loading and encapsulation efficiency of the No. 1 carvacrol stock solution emulsion and the No. 4 5% carvacrol sample emulsion, using the following methods:

[0072] The No. 1 carvacrol stock solution emulsion and the No. 4 5% carvacrol sample emulsion were separately dispersed in 2 mL of methanol, sonicated for 1 hour, and then filtered through a 0.22 μm filter membrane. The concentration of carvacrol in the samples was analyzed using a gas chromatography (GC) system equipped with a quartz capillary column HP-FFAP (30 m × 0.25 mm, 0.25 μm). GC conditions were as follows: injection temperature 250 °C, carrier gas He, column flow rate 1.0 mL / min, split ratio 80:1, and FID detector. Each experiment was performed in triplicate. Loading capacity (LC) and encapsulation efficiency (EE) were calculated using the following formulas:

[0073] Load capacity (LC):

[0074] ;

[0075] In the above formula, M represents the mass of the carvacrol stock solution and the 5% carvacrol sample added to the solution, and C represents the mass of the carvacrol sample added to the solution. car To determine the concentration of carvacrol in the solution, V Total To test the total volume of the solution.

[0076] Encapsulation efficiency (EE):

[0077] ;

[0078] In the above formula, C Total C represents the total amount of pesticide added to the solution. car The concentration at which carvacrol is released.

[0079] The calculation results show that the average encapsulation efficiency of the No. 1 carvacrol stock solution emulsion was 78.859%, and the average encapsulation efficiency of the No. 4 5% carvacrol sample emulsion was 54.263%. The average loading of the No. 1 carvacrol stock solution emulsion was 81.975%, and the average loading of the No. 4 5% carvacrol sample emulsion was 91.731%.

[0080] Further testing was conducted on the release rates of carvacrol stock solution emulsion No. 1 and carvacrol sample emulsion No. 4, using the following methods:

[0081] Take 5 mL of the prepared carvacrol stock solution emulsion and place it into a dialysis bag with a molecular weight cutoff of 1400. Place this dialysis bag in 50 mL of 50 vol% methanol solution, seal, and continuously stir. At room temperature, pipette 1 mL of the methanol solution at different time points, simultaneously adding the same volume of release medium to the solution system to maintain a constant volume. Prepare a standard solvent of known concentration, and pass the samples taken at different time points through a GC column to measure the characteristic peak areas. GC conditions: quartz capillary column HP-FFAP (30 m × 0.25 mm, 0.25 μm), injection temperature 250℃, carrier gas He, column flow rate 1.0 mL / min, split ratio 80:1, FID detector. The release rate of carvacrol at each time point was calculated using the external standard method.

[0082] Take 10 mL of the prepared 5% carvacrol sample emulsion (sample number 4) and place it in a dialysis bag with a molecular weight cutoff of 1400. Put this dialysis bag into 50 mL of 50% methanol solution, seal, and continuously stir. At room temperature, pipette 1 mL of the methanol solution at different time points, simultaneously adding the same volume of release medium to the solution system to maintain a constant volume. Prepare a standard solvent of known concentration, and pass the samples taken at different time points through a GC column to measure the characteristic peak areas. GC conditions: quartz capillary column HP-FFAP (30 m × 0.25 mm, 0.25 μm), injection temperature 250℃, carrier gas He, column flow rate 1.0 mL / min, split ratio 80:1, FID detector. The cumulative release rate of carvacrol at each time point was calculated using the external standard method.

[0083] Figure 9 The relationship between the cumulative release rate of carvacrol in the No. 1 carvacrol stock solution emulsion and time; Figure 10 The relationship between the cumulative release rate of carvacrol in the emulsion of 5% carvacrol sample No. 4 and time.

[0084] Figures 9-10 The results showed that the No. 1 carvacrol stock solution emulsion could achieve a release rate of 50% in about 30 minutes; the No. 4 5% carvacrol sample emulsion could achieve a release rate of 50% in about 720 minutes.

[0085] The effects of spraying No. 1 carvacrol stock solution emulsion and No. 4 5% carvacrol sample emulsion on the growth of citrus leaves were tested using the following methods:

[0086] After applying either No. 1 carvacrol stock solution emulsion or No. 4 5% carvacrol sample emulsion to citrus leaves, the growth of citrus leaves on the sprayed area, the unsprayed area on the same branch, and adjacent branches was observed at 1, 2, 3, 5, 7, 9, and 14 days. The yellowing, leaf drop, and wrinkling were observed. The control group was not sprayed with the emulsion.

[0087] Figure 11 Photos of citrus leaf morphology and growth status in the control group from day 1 to day 14; Figure 12 Photos of citrus leaf morphology and growth status 1–14 days after spraying No. 1 carvacrol stock solution emulsion; Figure 13 These are photos of the morphology and growth status of citrus leaves 1–14 days after spraying with 5% carvacrol sample emulsion No. 4.

[0088] from Figures 11-13 It can be seen that the citrus leaves on the sprayed parts, the unsprayed parts of the same branch, and the adjacent branches did not show yellowing, withering, or wrinkling 9 days or before. On the 14th day, the citrus leaves showed slight wrinkling as a whole, but not on the sprayed leaves. It is speculated that the wrinkling of the citrus leaves was caused by the environment.

[0089] The absorption of carvacrol stock solution emulsion No. 1 and carvacrol sample emulsion No. 4 by citrus leaves was tested using the following method:

[0090] Drop the sample emulsion onto citrus leaves and observe their contact angles.

[0091] Two mL of different samples were evenly spread onto pre-weighed citrus leaves, and then the emulsions were allowed to dry naturally under standard conditions (25°C). After drying, the total mass of the substrate was weighed again, and the residual mass of the emulsion was calculated to obtain the spreading properties of different samples.

[0092] The sample emulsion was dropped onto citrus leaves, and the adhesion of the emulsion was determined by observing how well it remained on the leaves.

[0093] Figure 14 The contact angle of carvacrol stock solution emulsion No. 1 dropped onto citrus leaves; Figure 15 The contact angle of sample emulsion No. 4 (5% carvacrol) dropped onto citrus leaves.

[0094] Figures 14-15 The results showed that the contact angles of carvacrol stock solution emulsion No. 1 on citrus leaves were 54.9° and 56.8°; while the contact angles of carvacrol sample No. 4 on citrus leaves were 23° and 25.5°. The results indicate that the carvacrol sample No. 4 emulsion is hydrophilic.

[0095] Figure 16 The results of the spreading properties test of different samples on citrus leaves. Figure 16In the preparation of the emulsions, Group 1 is the No. 1 carvacrol stock emulsion; Group 1, compared to the No. 1 carvacrol stock emulsion, omits the modified SiO2 nanoparticles during preparation; Group 4 is the No. 4 5% carvacrol sample emulsion; Group 4, compared to the No. 4 5% carvacrol sample emulsion, omits the modified SiO2 nanoparticles during preparation; and the nanomaterial group, compared to the No. 1 carvacrol stock emulsion, uses 10 mL of vegetable oil containing dissolved modified SiO2 nanoparticles as the oil phase during preparation. All test results at different time points are in mg.

[0096] Figure 16 The results showed that the emulsion group with modified SiO2 nanoparticles had better spreading properties than the solution group without modified SiO2 nanoparticles.

[0097] Figure 17 The results show the adhesion of carvacrol stock solution emulsion No. 1 and carvacrol sample emulsion No. 4 to citrus leaves.

[0098] from Figure 17 As can be seen, when the No. 1 carvacrol stock solution emulsion or the No. 4 5% carvacrol sample emulsion was dropped onto the citrus leaves, it quickly flowed down the leaves, indicating that its adhesion was poor.

[0099] The distribution of the nanomaterial emulsion sample prepared in the spreading test in citrus leaves and stems was determined using the following method:

[0100] Cy5 fluorescently labeled nanomaterial emulsions were prepared. After spraying the emulsions, leaves sprayed with the emulsion, unsprayed parts of the same branch, and adjacent leaves of different citrus plants were collected at 2, 4, 6, 8, 10, and 24 hours. The emulsions were ground into homogenates and their fluorescence intensity was measured, with free Cy5 as a control. Citrus stems sprayed with the emulsions were collected at 2, 4, 6, 8, and 10 hours. The emulsions were ground into homogenates and their fluorescence intensity was measured, with free Cy5 (the addition of modified SiO2 nanoparticles was omitted compared to the nanomaterial emulsions) as a control.

[0101] Figure 18 The fluorescence values ​​of the sample emulsion of Cy5 fluorescently labeled nanomaterials and the citrus leaves and adjacent leaves after spraying with free Cy5 were measured. Figure 19 Fluorescence values ​​of citrus stems sprayed with Cy5-labeled 5% carvacrol sample emulsion and free Cy5.

[0102] Figures 18-19The results showed that a stronger signal was detected in the fluorescently labeled emulsion group in the tissue homogenate. After 8 hours, the fluorescence value of citrus leaves on the same branch was higher than that of adjacent plants and the group sprayed with nanomaterial emulsion. Simultaneously, stem homogenate analysis revealed varying fluorescence intensity over time. Comparing the free fluorophore group and the fluorescently labeled emulsion group, the fluorescence detected within the plant was stronger and more widely distributed. The varying stem fluorescence values ​​over time demonstrate that the emulsion entered the leaves, and the addition of modified SiO2 nanoparticles facilitated the delivery of the emulsion into the citrus leaves. The distribution of the modified SiO2 nanoparticles was observed on adjacent leaves of different plants after spraying.

[0103] The absorption of carvacrol stock solution emulsion No. 1 and carvacrol sample emulsion No. 4 by citrus leaves was tested using the following method:

[0104] Method 1: Spray the No. 1 carvacrol stock solution emulsion or the No. 4 5% carvacrol sample emulsion onto citrus leaves and fruits. Collect citrus leaves at 2, 4, 6, 8, 10, and 24 hours. Weigh 100 mg of citrus leaf sample into a 1 mL centrifuge tube, add 2 mL of acetonitrile, and sonicate for 20 min. Add 40 mg of sodium chloride, vortex for 5 min, centrifuge at 6000 r / min for 5 min, and transfer 1 mL of the supernatant to a 2 mL centrifuge tube containing 50 mg of PSA. Vortex for 1 min, filter through a 0.22 μm filter membrane, and perform GC analysis.

[0105] Method 2: Spray the No. 1 carvacrol stock solution emulsion or the No. 4 5% carvacrol sample emulsion onto citrus leaves and fruits. Collect citrus leaves at 2, 4, 6, 8, 10, and 24 hours. Weigh 100 mg of citrus leaf sample into a 1 mL centrifuge tube, add 1 mL of 1% acetic acid-acetonitrile, and extract ultrasonically for 25 min. Add 100 mg of anhydrous MgSO4 and 50 mg of NaCl, shake vigorously for 1 min, and then centrifuge at 10000 r / min for 5 min in a refrigerated centrifuge. Transfer 2 mL of the supernatant after centrifugation to an ep tube containing 40 mg of PSA, vortex for 1 min, and centrifuge at 3000 r / min for 5 min. Filter the supernatant through a 0.22 μm filter membrane and perform GC analysis.

[0106] Method 3: Spray the No. 1 carvacrol stock solution emulsion or the No. 4 5% carvacrol sample emulsion onto citrus leaves and fruits. Collect citrus leaves at 2, 4, 6, 8, 10, and 24 hours. Weigh 1.5g of citrus leaf sample into a 10mL centrifuge tube, add 10mL of acetonitrile, and extract using ultrasound for 30min three times; add 10mL of methanol, and extract using ultrasound for 30min three times. Vigorously shake with 1000mg NaCl for 1min, then vortex for 1min. Concentrate the solution 6 times, transfer 1mL of the supernatant after centrifugation into an EP tube containing 40mg PSA, and let stand overnight; filter the supernatant through a 0.22μm filter membrane and perform GC analysis.

[0107] The results showed that carvacrol was not detected by any of the three sampling and extraction methods. Even after large-scale spraying, subsequent sampling, extraction, and concentration yielded no characteristic peaks for carvacrol. Simultaneously, extracts from adjacent citrus leaves at two and ten hours, as well as from adjacent leaves of different citrus trees, also showed no characteristic peaks for carvacrol. The absence of detection on sprayed citrus leaves suggests that the nano-combination in the emulsion likely provides a slow-release effect, and the issue of it entering the leaves probably stemmed from evaporation over time. Furthermore, the lack of detection at two and ten hours indicates that no carvacrol residue remained.

[0108] The effect of carvacrol stock solution emulsion No. 1 on the bioactivity of citrus psyllids was tested using the following method:

[0109] The carvacrol stock solution emulsion was diluted before use (to an effective carvacrol concentration of 0.07%). The carvacrol technical grade was first prepared as a 0.7 wt% stock solution, then diluted 10 times before use. Three treatment groups were conducted (pure water, 0.07% carvacrol technical grade, and diluted sample emulsion containing 0.07% carvacrol), with three replicates per group. The solutions from different groups were added to a sprayer and sprayed 15 times onto citrus seedlings containing 10 citrus psyllids in a net cup, for a total volume of approximately 1.6 mL. The number of citrus psyllid deaths within 48 hours was recorded.

[0110] Figure 20 Mortality rates of citrus psyllids in different treatment groups; Figure 21 The distribution of citrus psyllids on citrus seedlings after 48 hours in different treatment groups.

[0111] from Figures 20-21 As can be seen, water, as a control treatment, had no effect on the growth of citrus psyllids. Treatment with carvacrol technical grade (0.07%) resulted in a low mortality rate for citrus psyllids, reaching 10% after 48 hours. Spraying with carvacrol technical grade emulsion (0.07% effective carvacrol concentration) resulted in a 93% mortality rate within a short period (6 hours), reaching 100% after 48 hours. Due to the low concentration of carvacrol and its volatile nature, the carvacrol technical grade had low toxicity to citrus psyllids, resulting in a low mortality rate. Surviving psyllids showed good growth and were distributed on the leaves for feeding. The sample emulsion provided some protection against carvacrol and promoted its entry into the leaves. Therefore, the carvacrol complex encapsulated in the emulsion achieved a good insecticidal effect within a short treatment period (6 hours), and the insecticidal effect reached 100% with prolonged treatment time.

[0112] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a Pickering emulsion loaded with carvacrol, characterized in that, Includes the following steps: First, cholesterol is grafted onto sodium alginate to obtain cholesterol-grafted sodium alginate. Then, the cholesterol-grafted sodium alginate and SiO2 nanoparticles are co-dispersed in water, allowing the cholesterol-grafted sodium alginate to adsorb onto the SiO2 nanoparticles, resulting in modified SiO2 nanoparticles. The modified SiO2 nanoparticles and carvacrol are added to the oil phase and combined with the aqueous phase to prepare a Pickering emulsion, which is the carvacrol-loaded Pickering emulsion.

2. The method for preparing the Pickering emulsion loaded with carvacrol according to claim 1, characterized in that, The preparation steps of the cholesterol-grafted sodium alginate include: dissolving sodium alginate and anhydrous p-toluenesulfonic acid together in 38 mL of formic acid / N,N-dimethylformamide mixed solvent, and stirring at 55 °C for 30 min to protonate; then adding 4-dimethylaminopyridine, N,N-dicyclohexylcarbodiimide and cholesterol dissolved in chloroform to the system, and reacting at 40 °C for 24 h to obtain cholesterol-grafted sodium alginate.

3. The method for preparing the Pickering emulsion loaded with carvacrol according to claim 2, characterized in that, The ratio of sodium alginate, anhydrous p-toluenesulfonic acid, formic acid / N,N-dimethylformamide mixed solvent, 4-dimethylaminopyridine, N,N-dicyclohexylcarbodiimide, and cholesterol is 1.0g:0.324g:38mL:0.475g:0.4g:1.0g; the volume ratio of formic acid to N,N-dimethylformamide in the formic acid / N,N-dimethylformamide mixed solvent is 10:

9.

4. The method for preparing the Pickering emulsion loaded with carvacrol according to claim 1, characterized in that, The mass ratio of cholesterol-grafted sodium alginate to SiO2 nanoparticles is 1:

5.

5. The method for preparing the Pickering emulsion loaded with carvacrol according to claim 1, characterized in that, The process of adsorbing cholesterol-grafted sodium alginate onto SiO2 nanoparticles also includes a treatment step to reduce the particle size of the modified SiO2 nanoparticles.

6. The method for preparing the Pickering emulsion loaded with carvacrol according to claim 1, characterized in that, The carvacrol is carvacrol stock solution or carvacrol-containing emulsion.

7. The method for preparing the Pickering emulsion loaded with carvacrol according to claim 1, characterized in that, The concentration of modified SiO2 nanoparticles in the oil phase is 10 mg / mL.

8. A Pickering emulsion loaded with carvacrol prepared by the method of preparing the Pickering emulsion loaded with carvacrol according to any one of claims 1 to 7.

9. The use of the Pickering emulsion loaded with carvacrol as described in claim 8 in the preparation of an agent for controlling citrus psyllids.

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