Preparation method and application of nanometer liposome

Abstract

This invention discloses a method for preparing and applying nanoliposomes, relating to the fields of kiwifruit soft rot control and kiwifruit preservation. The preparation method includes the following steps: mixing thymol, cholesterol, and oleic acid to obtain an oil phase; mixing polyethylene glycol, lauryl ether phosphate, an emulsifying dispersant, and water to obtain an aqueous phase; mixing the oil phase and the aqueous phase, performing high-speed emulsification and shearing followed by low-speed stirring to obtain nanoliposomes. This invention uses cholesterol and oleic acid as wall materials and polyethylene glycol as a modifying material to successfully load thymol into nanoliposomes via microemulsification, achieving highly efficient encapsulation of thymol. The obtained thymol-containing nanoliposomes exhibit excellent performance and can be used to prepare formulations for controlling kiwifruit soft rot, demonstrating excellent control effects against the disease.

Description

Technical Field

[0001] This invention relates to the fields of kiwifruit soft rot prevention and kiwifruit preservation technology, and in particular to a method for preparing and applying nanoliposomes. Background Technology

[0002] Kiwifruit soft rot is one of the most destructive diseases affecting kiwifruit during post-harvest storage, transportation, and sales. It is not caused by a single pathogen, but rather by a combination of multiple fungal pathogens leading to fruit rot. Its main characteristic is a rapid outbreak after the fruit has softened during ripening, causing the flesh to soften, rot, and spoil, resulting in significant economic losses. The main pathogens causing kiwifruit soft rot include *Botrytis cinerea* (…). Botryosphaeria dothidea ), Pseudostem mold ( Phomopsis spp. ), Botrytis cinerea ( Botrytis cinerea (i.e., gray mold) and Penicillium ( Penicillium spp. )wait.

[0003] Currently, the global control of kiwifruit soft rot generally follows the principle of "prevention first, comprehensive management." However, current control efforts still heavily rely on chemical control methods, primarily using copper-based fungicides, triazoles, and methoxyacrylates, in the form of wettable powders, emulsifiable concentrates, and aqueous solutions. These methods have the following drawbacks: While copper-based fungicides are effective broad-spectrum insecticides, their harmful effects cannot be ignored. Firstly, copper-based fungicides can easily cause severe phytotoxicity in high temperature and humidity or during sensitive crop stages (such as young fruit and seedling stages), leading to leaf burn, leaf and fruit drop. Secondly, copper, as a heavy metal, is difficult to degrade; long-term use can cause copper accumulation in the soil, disrupting soil microbial ecology, leading to compaction and acidification, and potentially polluting waterways through rainwater runoff, posing a high toxicity to fish and other aquatic organisms. Improper use can also pose potential health risks to humans through residues or direct contact. Furthermore, long-term application of copper-based fungicides leads to high resistance in pathogens, reducing their effectiveness in controlling kiwifruit soft rot. Triazoles and methoxyacrylates, among other chemical fungicides, have relatively concentrated sites of action, making pathogens prone to developing resistance and gradually reducing their control efficiency. Furthermore, they easily cause crop damage in hot and humid environments, disrupt field microecology, and exacerbate the expansion of harmful organisms such as mites. Currently, commonly used chemical fungicides are formulated as wettable powders, emulsifiable concentrates, and aqueous solutions, but their systemic conductivity is weak, especially in penetrating the phloem, and they have poor rain resistance, thus limiting their effectiveness in areas with frequent rainfall. Some systemic synthetic fungicides also pose risks of pesticide residues and resistance development, and their compatibility with alkaline substances is limited, restricting their application scope. Against this backdrop, developing novel fungicides with new mechanisms of action, high-efficiency fungicidal activity, and good environmental compatibility has become a key research focus. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing and applying nanoliposomes to address the problems of poor efficacy, phytotoxicity, and adverse environmental impacts associated with existing chemical agents used to control kiwifruit soft rot. The core of this invention lies in preparing nanoliposomes encapsulated with thymol via microemulsion, thereby achieving highly efficient and environmentally friendly control of kiwifruit soft rot.

[0005] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of the present invention: a method for preparing nanoliposomes, comprising the following steps: Thymol, cholesterol, and oleic acid are mixed to obtain an oil phase; polyethylene glycol, lauryl ether phosphate, emulsifying dispersant, and water are mixed to obtain an aqueous phase; the oil phase and the aqueous phase are mixed, emulsified and sheared at high speed, and then stirred at low speed to obtain the nanoliposomes (specifically, nanoliposomes encapsulating thymol, i.e., thymol nanoliposomes).

[0006] This invention addresses the problems of poor efficacy and adverse environmental impact of chemical agents in controlling kiwifruit soft rot by proposing a highly efficient, green, and environmentally friendly thymol nanoliposome formulation for the prevention and control of kiwifruit soft rot. Thymol is a naturally occurring monoterpenoid compound, mainly found in the essential oils of plants such as thyme and oregano. It possesses a fresh, rich herbal aroma and significant antibacterial and antifungal properties. This invention selects thymol, a natural fungicide, as the active ingredient for controlling kiwifruit soft rot, offering advantages such as multi-target action, easy degradation, and low ecotoxicity.

[0007] However, thymol is highly volatile and easily oxidized, leading to degradation or inactivation of the drug components when used directly, thus reducing its effective utilization rate. To address this issue, this invention formulates thymol into nanoliposomes, avoiding degradation caused by volatilization and ultraviolet radiation, thereby improving the utilization rate of thymol. With its nanoscale particle size and excellent lipophilicity, nanoliposomes can efficiently penetrate cell and tissue barriers, achieving effective delivery within the body, thus precisely delivering the active ingredient thymol to the target location. This not only significantly improves the bioavailability of the active ingredient thymol but also enhances the control effect against soft rot disease in kiwifruit.

[0008] The choice of liposome wall material is highly correlated with the encapsulated drug component, directly affecting whether the drug component can be fully encapsulated. In this invention, oleic acid and cholesterol are used as wall materials, which offer advantages over other wall materials in terms of morphology, particle size, encapsulation performance, and safety. Furthermore, this invention uses polyethylene glycol (PEG) to modify the nanoliposomes. PEG modification improves the mechanical strength of the nanoliposomes and effectively prevents adhesion and aggregation. This results in nanoliposomes with regular morphology, no agglomeration, and smaller particle size. The thymol nanoliposomes prepared using oleic acid and cholesterol as wall materials and modified with PEG were characterized by transmission electron microscopy, dynamic light scattering, controlled-release performance testing, in vitro bioassay, and fruit preservation experiments. The liposomes are spherical with an average particle size of 190.5 nm. The sustained-release assay results show that the thymol in the nanoliposomes is released continuously for 2-3 days, with a cumulative release rate of 80.03%.

[0009] In addition, in terms of preparation technology, this invention uses microemulsion to prepare nanoliposomes. Compared with traditional liposome preparation techniques (such as thin film method), microemulsion does not require the use of harmful organic solvents (such as ethanol, dichloromethane and other harmful organic solvents), which is safer for mammals and the environment, has environmentally friendly characteristics, and exhibits excellent environmental performance.

[0010] Furthermore, the mass ratio of thymol, cholesterol, and oleic acid is 2:2:4.

[0011] Furthermore, the mass ratio of polyethylene glycol, lauryl ether phosphate, emulsifying dispersant, and water is 10:2:2:78.

[0012] Furthermore, the mass ratio of cholesterol in the oil phase to polyethylene glycol in the aqueous phase is 2:10.

[0013] The ratio of active ingredient to wall material also affects the encapsulation effect of active ingredient. In this invention, the ratio of thymol to wall material is the optimal ratio. Too high or too low a ratio will result in the drug ingredient not being fully encapsulated.

[0014] Preferably, the average molecular weight of the polyethylene glycol is 1000-20000.

[0015] More preferably, the polyethylene glycol is PEG4000.

[0016] Preferably, the emulsifying dispersant is a fatty alcohol polyoxyethylene ether phosphate emulsifying dispersant, including model 5218CP.

[0017] Furthermore, after mixing thymol, cholesterol, and oleic acid, the process also includes a heating and stirring step; the heating and stirring temperature is 110-130℃, the rotation speed is 600-1000rpm, and the time is 0.5-1h.

[0018] Furthermore, after mixing polyethylene glycol, lauryl ether phosphate, emulsifying dispersant and water, the process also includes a heating and stirring step; the heating and stirring temperature is 110-130℃, the rotation speed is 600-1000rpm, and the time is 0.5-1h.

[0019] Furthermore, the high-speed emulsification shearing rotation speed is 6000-10000 rpm, and the time is 3-10 min.

[0020] Furthermore, the low-speed stirring speed is 500-800 rpm, and the time is 0.5-2 h.

[0021] Furthermore, the high-speed emulsification shearing is carried out at 110-130°C.

[0022] Furthermore, the low-speed stirring is carried out at room temperature.

[0023] The second technical solution of the present invention: a nanoliposome prepared by the above-described method for preparing nanoliposomes.

[0024] The third technical solution of the present invention: the application of the above-mentioned nanoliposomes in the preparation of a formulation for preventing and treating soft rot of kiwifruit.

[0025] The fourth technical solution of the present invention: a preparation for preventing and controlling soft rot of kiwifruit, the raw materials of which include the above-mentioned nanoliposomes.

[0026] The fifth technical solution of the present invention: the application of the above-mentioned nanoliposomes or the above-mentioned preparations for preventing and controlling soft rot of kiwifruit in the prevention and control of soft rot of kiwifruit.

[0027] Furthermore, the application methods include: during the fruit enlargement period of kiwifruit until harvest, using the nanoliposomes or the preparation for preventing and controlling kiwifruit soft rot, diluted at a certain dilution ratio (400-600 times), and spraying the entire kiwifruit plant with 1-1.5 L of the agent per plant (diluted nanoliposomes or the preparation for preventing and controlling kiwifruit soft rot), focusing on spraying the fruit; or after kiwifruit harvest, diluting the nanoliposomes or the preparation for preventing and controlling kiwifruit soft rot and soaking the kiwifruit fruit.

[0028] This invention utilizes cholesterol and oleic acid as wall materials and polyethylene glycol as a modifying material to successfully load thymol into nanoliposomes via microemulsion, achieving highly efficient encapsulation of thymol and obtaining a high-performance thymol nanoliposome formulation (small particle size, uniform dispersion, high bioavailability, and sustained-release effect). Using a Hitachi HT-7800 transmission electron microscope, the thymol nanoliposomes were observed to have a smooth, rounded surface without collapse, good encapsulation, a thick outer shell, and good mechanical strength. In in vitro bioassays, compared to other essential oils, thymol showed a significant inhibitory effect on soft rot in kiwifruit. The EC50 of the thymol nanoliposomes was [missing data]. 50 The concentration was only 43.865 mg / L. In fruit preservation trials, compared to traditional pesticides, thymol nanoliposomes effectively inhibited kiwifruit soft rot infection, significantly reduced the incidence of kiwifruit disease, and slowed the spread of lesions. This invention provides an innovative solution for the prevention and control of kiwifruit soft rot.

[0029] Furthermore, to verify the advantages of the present invention's technical solution of using oleic acid and cholesterol as wall materials and modifying them with polyethylene glycol (PEG), the present invention systematically compared the effects of different wall material systems on the performance of thymol nanoliposomes. Experimental results showed that compared to the traditional soybean lecithin / cholesterol system (encapsulation efficiency 68.5%, particle size 320.7 nm) or the single oleic acid system (encapsulation efficiency 72.1%), the use of an oleic acid and cholesterol composite wall material significantly improved the encapsulation efficiency and reduced the particle size. Among these, the system with an oleic acid to cholesterol ratio of 4:2 and PEG modification achieved the best overall performance: an encapsulation efficiency as high as 82.7%, an average particle size of only 190.5 nm, and uniform distribution and system stability. This indicates that this specific ratio of oleic acid to cholesterol achieves the optimal balance between membrane stability and drug-containing space, while PEG modification effectively prevents particle aggregation through steric hindrance, thus synergistically obtaining highly encapsulated, small-particle-size, and stable nanoliposomes, laying a solid material foundation for the efficient control of soft rot in kiwifruit using these nanoliposomes.

[0030] The present invention discloses the following technical effects: The thymol nanoliposomes of the present invention have the following advantages over existing formulations: (1) Nanoscale size: Thymol nanoliposomes were prepared by microemulsion. The nanoscale size can give the formulation strong permeability, making it easier for the effective active ingredient thymol to reach the target site and improve the utilization rate of the effective active ingredient. (2) Lipophilic properties: Due to the excellent lipophilicity of nanoliposomes, they can effectively penetrate the cell tissue barrier and achieve efficient conduction in plants; (3) Good stability: The use of polyethylene glycol (PEG) to modify liposomes improves the mechanical strength of the nanoliposomes and effectively prevents liposome adhesion and aggregation. Combined with a unique dispersing surfactant combination, the thymol nanoliposomes exhibit good stability and longer retention. Specifically, the PEG chain can significantly increase the flexural modulus of the liposome membrane through steric hindrance. Electron microscopy observation shows that the vesicle shells of PEG-modified liposomes are thicker and denser. In the stability comparison experiment, the unmodified liposomes showed obvious aggregation after storage due to insufficient structural strength, and the average particle size increased significantly; while the PEG-modified liposomes of this invention maintained stable particle size and system uniformity under the same conditions. This directly proves that PEG modification effectively improves the mechanical strength and physical stability of liposomes by enhancing their structural integrity.

[0031] (4) Safety: This invention prepares thymol nanoliposomes by microemulsion, without the use of harmful organic solvents. While effectively preventing and controlling soft rot of kiwifruit, it reduces the adverse impact on the environment and meets the production standards of green agents and green food. Attached Figure Description

[0032] 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.

[0033] Figure 1 The images shown are TEM images of thymol nanoliposomes prepared in Example 1 and Comparative Example 1, where A and B are TEM images of Example 1, and C and D are TEM images of Comparative Example 1.

[0034] Figure 2 The particle size distribution diagram is shown for the thymol nanoliposomes prepared in Example 1.

[0035] Figure 3 The effects of different agents on the inhibition of soft rot pathogens in kiwifruit.

[0036] Figure 4 The results show the sustained-release performance of thymol nanoliposomes, where A is the sustained-release curve of thymol nanoliposomes and B is the fitting analysis result of the release curve.

[0037] Figure 5 The rotting of kiwifruit after treatment with different concentrations of carbendazim and thymol nanoliposomes.

[0038] Figure 6The rotting condition of kiwifruit after treatment with thymol nanoliposomes and thymol emulsion. 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.

[0040] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to 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, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0041] 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 or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0042] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[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] 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.

[0045] In the following embodiments, comparative examples and test examples of the present invention, if room temperature is involved, it specifically refers to 25±2℃.

[0046] All raw materials used in the following examples, comparative examples and test examples of this invention are commercially available products. Among them, the emulsifying dispersant 5218CP was purchased from Shanghai Xingfei Chemical Co., Ltd.; and the polyethylene glycol was PEG4000.

[0047] Example 1 A method for preparing thymol nanoliposomes, comprising the following steps: 2g of thymol and 2g of cholesterol were added to 4g of oleic acid and heated and stirred at 800rpm for 0.5h at 120℃ on a magnetic stirrer to fully dissolve the thymol and cholesterol, obtaining the oil phase. 10g of polyethylene glycol was added to 78g of deionized water, followed by 2g of AEO-3P and 2g of emulsifying dispersant 5218CP. The mixture was heated and stirred at 800rpm for 0.5h at 120℃ on a magnetic stirrer to obtain the aqueous phase. The oil phase was poured into the aqueous phase, and emulsified and sheared at 8000rpm for 5min at 120℃ using a T25 digital display disperser to obtain a stable oil-in-water emulsion. Then, the mixture was stirred at 600rpm for 1h at room temperature on a magnetic stirrer to obtain a thymol nanoliposome solution.

[0048] Comparative Example 1 A method for preparing thymol nanoliposomes, comprising the following steps: 2g of thymol and 2g of cholesterol were added to 4g of oleic acid and heated and stirred at 800rpm for 0.5h at 120℃ on a magnetic stirrer to fully dissolve the thymol and cholesterol, obtaining the oil phase. 2g of AEO-3P and 2g of emulsifying dispersant 5218CP were added to 88g of deionized water and heated and stirred at 800rpm for 0.5h at 120℃ on a magnetic stirrer, obtaining the aqueous phase. The oil phase was poured into the aqueous phase, and emulsified and sheared at 8000rpm for 5min at 120℃ using a T25 digital display disperser to obtain a stable oil-in-water emulsion. Then, the mixture was stirred at 600rpm for 1h at room temperature on a magnetic stirrer to obtain a thymol nanoliposome solution.

[0049] Comparative Example 2 6g of soybean lecithin was added to 20g of dichloromethane and stirred at 800rpm at room temperature on a magnetic stirrer until the soybean lecithin was completely dissolved in the dichloromethane. Then, 2g of thymol was added, and stirring was continued at 800rpm at room temperature until fully dissolved, yielding the oil phase. 88g of deionized water was weighed, and 2g of AEO-3P and 2g of 5218CP were added. The mixture was heated and stirred at 800rpm at room temperature for 0.5h on a magnetic stirrer, yielding the aqueous phase. The oil phase was poured into the aqueous phase, and emulsified and sheared at 8000rpm for 5min at room temperature using a T25 digital display disperser, yielding a stable oil-in-water emulsion. The emulsion was then stirred at 600rpm at room temperature for 6h on a magnetic stirrer to remove the organic solvent, yielding a thymol nanoliposome solution.

[0050] Comparative Example 3 2g cholesterol and 4g soy lecithin were added to 20g dichloromethane. The mixture was stirred at 800 rpm at room temperature using a magnetic stirrer until the soy lecithin was completely dissolved. Then, 2g thymol was added, and the mixture was stirred at 800 rpm at room temperature until fully dissolved, yielding the oil phase. 88g deionized water was weighed, and 2g AEO-3P and 2g 5218CP were added. The mixture was stirred at 800 rpm at room temperature for 0.5 hours using a magnetic stirrer, yielding the aqueous phase. The oil phase was poured into the aqueous phase, and the mixture was emulsified and sheared at 8000 rpm for 5 minutes at room temperature using a T25 digital display disperser, yielding a stable oil-in-water emulsion. The emulsion was then stirred at 600 rpm at room temperature for 6 hours using a magnetic stirrer to remove the organic solvent, yielding a thymol nanoliposome solution.

[0051] Comparative Example 4 Same as Example 1, except that cholesterol and other substances are replaced with oleic acid (i.e., the wall material is only oleic acid, and the amount of oleic acid used is 6g).

[0052] Comparative Example 5 6g of cholesterol was added to 20g of dichloromethane and stirred at 800rpm at room temperature on a magnetic stirrer until the soybean lecithin was completely dissolved in the dichloromethane. Then, 2g of thymol was added, and stirring was continued at 800rpm at room temperature until fully dissolved, yielding the oil phase. 88g of deionized water was weighed, and 2g of AEO-3P and 2g of 5218CP were added. The mixture was stirred at 800rpm at room temperature for 0.5h on a magnetic stirrer, yielding the aqueous phase. The oil phase was poured into the aqueous phase, and emulsified and sheared at 8000rpm for 5min at room temperature using a T25 digital display disperser, yielding a stable oil-in-water emulsion. The emulsion was then stirred at 600rpm at room temperature for 6h on a magnetic stirrer to remove the organic solvent, yielding a thymol nanoliposome solution.

[0053] Comparative Example 6 Same as Example 1, except that the amount of oleic acid is adjusted to 5g and the amount of cholesterol is adjusted to 1g.

[0054] Comparative Example 7 Same as Example 1, except that AEO-3P is replaced with Tween 80.

[0055] Comparative Example 8 4g of oleic acid, 2g of cholesterol, and 2g of thymol were added to a mixed solvent of dichloromethane and ethanol (30mL dichloromethane + 10mL ethanol). After mixing thoroughly, the solvent was removed by rotary evaporation under reduced pressure, allowing the lipids to form a uniform film on the inner wall of the flask. Subsequently, 92g of deionized water was added to the film to fully hydrate and disperse it to form multilayer liposomes. Finally, the resulting suspension was sonicated (800W for 25min) to reduce the particle size and improve the uniformity of dispersion, resulting in a thymol nanoliposome solution.

[0056] Comparative Example 9 Same as Example 1, except that the preparation steps of the aqueous phase are adjusted as follows: 5g of polyethylene glycol is added to 83g of deionized water, then 2g of AEO-3P and 2g of emulsifying dispersant 5218CP are added, and the mixture is heated and stirred at 800rpm for 0.5h at 60°C on a magnetic stirrer to obtain the aqueous phase.

[0057] Comparative Example 10 Same as Example 1, except that the preparation steps of the aqueous phase are adjusted as follows: 15g of polyethylene glycol is added to 73g of deionized water, then 2g of AEO-3P and 2g of emulsifying dispersant 5218CP are added, and the mixture is heated and stirred at 800rpm for 0.5h at 60°C on a magnetic stirrer to obtain the aqueous phase.

[0058] Comparative Example 11 Same as Example 1, except that the amount of thymol was adjusted to 1g and the amount of deionized water was adjusted to 79g.

[0059] Comparative Example 12 Same as Example 1, except that the amount of thymol was adjusted to 3g and the amount of deionized water was adjusted to 77g.

[0060] Test Example 1 Morphological characterization, physicochemical properties and stability testing: (1) Observation of lipid in vitro morphology The morphological characteristics and adhesion of thymol nanoliposomes were observed using a Hitachi HT-7800 transmission electron microscope (TEM) from Japan.

[0061] Figure 1The images show TEM images of thymol nanoliposomes prepared in Example 1 and Comparative Example 1, where A and B are TEM images of Example 1, and C and D are TEM images of Comparative Example 1. It can be seen that the thymol nanoliposomes prepared in both Example 1 and Comparative Example 1 exhibit a spherical structure with a smooth and rounded surface, dense capsule walls without collapse, and good capsule formation rate. However, in Example 1, polyethylene glycol was used. Polyethylene glycol modified the liposome shell, resulting in a moderate shell thickness and improved liposome dispersibility, effectively inhibiting particle aggregation and adhesion. In contrast, the liposomes in Comparative Example 1, without polyethylene glycol modification, exhibited severe aggregation and adhesion.

[0062] (2) Particle size test The particle size of thymol nanoliposomes was tested by dynamic light scattering using a nanoparticle size analyzer.

[0063] Figure 2 The particle size distribution diagram of the thymol nanoliposomes prepared in Example 1 shows that the average particle size of the thymol nanoliposomes is 190.5 nm, and the polydispersity index (PDI, reflecting the uniformity of particle size distribution) is 0.18. The average particle size and PDI test results of each comparative example are shown in Table 1.

[0064] (3) Zeta potential Zeta potential tests were performed on the thymol nanoliposomes prepared in each example and comparative example to reflect the electrostatic stability of the thymol nanoliposomes. The results are shown in Table 1.

[0065] (4) Encapsulation efficiency test The encapsulation efficiency of the thymol nanoliposomes prepared in each example and comparative example was tested using the following methods: 1) Construction of the thymol standard curve: The thymol standard curve was constructed using a Waters e2695 high-performance liquid chromatograph (WATERS, USA). 10 mg of thymol standard was accurately weighed, dissolved and mixed with acetonitrile, and diluted to 100 mL to obtain a standard solution with a concentration of 100 mg / L. An appropriate amount of the thymol standard solution was accurately transferred and diluted with acetonitrile to prepare standard solutions with concentration gradients of 1 mg / L, 10 mg / L, 20 mg / L, 40 mg / L, 60 mg / L, 80 mg / L, and 100 mg / L. After the standard solutions were loaded into HPLC detection vials, the thymol standard curve was constructed using HPLC. The chromatographic conditions used in the experiment were as follows: Waters TC-C18 stainless steel column (4.6 mm × 250 mm, 5 μm), mobile phase V... 乙腈 V 水 =60:40, flow rate 1.0 mL / min, detection wavelength: 275 nm, injection volume: 10 μL, column temperature: 30 ℃.

[0066] Test of the encapsulation efficiency of thymol in nanoliposome solution (indirectly determined by measuring the amount of unencapsulated thymol): Freshly prepared nanoliposome solution was centrifuged at 13,000 rpm for 1 h at 4 °C using a 5804R high-speed benchtop centrifuge (Eppendorf, Germany). Sodium chloride solution was added to the nanoliposome solution to break the emulsion before centrifugation. The supernatant was then collected, filtered through a Millipore VR membrane (0.22 μm), and the amount of unencapsulated drug was determined by high-performance liquid chromatography (HPLC). The encapsulation efficiency was calculated according to formula (1), and the results are shown in Table 1.

[0067] Encapsulation rate = (total drug - amount of unencapsulated drug) / total drug × 100% (1).

[0068] (5) Stability test The thymol nanoliposome solutions prepared in each example and comparative example were sealed and stored at room temperature for 30 days. The average particle size was measured and compared with the average particle size before storage. The stability was characterized by the particle size growth rate. The results are shown in Table 1.

[0069] Table 1 Comparison of physicochemical properties and stability (3 samples were tested for each type of sample, and the results are expressed as mean ± standard deviation) As shown in Table 1, except for Comparative Example 11, the physicochemical properties and stability of each comparative example are not as good as those of Example 1. Although the encapsulation rate of Comparative Example 11 is higher than that of Example 1, the amount of drug added is low, the overall active ingredient content of the formulation is insufficient, and it is uneconomical.

[0070] Test Example 2 Antibacterial performance test: To determine the inhibitory effect of the active ingredient on soft rot of kiwifruit, an indoor bioactivity assay was conducted using the mycelial growth rate method (i.e., indoor bioassay), as detailed below: (1) Preparation of PDA culture medium: First, take 200g of peeled potato chunks, boil them, filter them with gauze, add 20g of glucose and 20g of agar, and then add distilled water until the total volume reaches 1000mL. Adjust the pH to 7.4 using NaOH. Dispense the culture medium into Erlenmeyer flasks and seal them tightly with sealing film. Then place them in an autoclave and sterilize at 121℃ for 20 minutes. After sterilization, turn off the power and wait for the pressure to drop to zero and the temperature to drop below 80℃. Then open the lid and take out the culture medium. When it cools to 50℃, pour it into plates to obtain PDA plate solid culture medium for later use.

[0071] (2) Collection of biological test materials: The pathogens causing soft rot of kiwifruit were collected, isolated and identified.

[0072] 1) Collection: Select typical soft rot diseased kiwifruit and cut the internal tissue at the junction of diseased and healthy tissue under aseptic conditions.

[0073] 2) Separation: The excised internal tissue was disinfected sequentially with 75wt% ethanol solution (30 seconds) and 1wt% sodium hypochlorite solution (2 minutes), rinsed 3 times with sterile water, and then placed on a PDA plate and incubated upside down in the dark at 26°C for 5 days.

[0074] 3) Purification: Pick the edge hyphae tips of newly grown colonies, transfer them to a new PDA plate, and purify to obtain single colonies.

[0075] 4) Identification: The pathogen was identified as Staphylococcus aureus through colony morphology, microscopic features and sequence molecular identification.

[0076] (3) Preparation of mycelium cake: The standard mycelium cake method is adopted. A sterile punch with a diameter of 5 mm is used to cut the mycelium cake from the edge of the cultured colony.

[0077] (4) Preparation of test reagents: Weigh 94g of deionized water, add 2g of AEO-3P and 2g of 5218CP emulsifying dispersant, and then add 2g of active ingredients (cinnamaldehyde, carvacrol, Litsea cubeba oil, thymol). Use a T25 digital display disperser to emulsify and shear at 8000rpm for 5min at room temperature to obtain a variety of stable water-in-oil mixed emulsions (i.e., water emulsions with a concentration of 2wt%) containing different active ingredients.

[0078] (5) Indoor bioactivity determination: The mycelial growth rate method was used. First, drug-containing plates were prepared: Under aseptic conditions, appropriate amounts of the 2wt% cinnamaldehyde emulsion, 2wt% carvacrol emulsion, 2wt% litsea cubeba oil emulsion, 2wt% thymol emulsion and 2wt% thymol nanoliposome solution (prepared in Example 1) prepared in step (4) were added to sterilized PDA medium cooled to 50℃. After thorough mixing, the plates were poured to prepare drug-containing plates with final concentrations of 16mg / L, 24mg / L, 32mg / L, 40mg / L, 48mg / L and 56mg / L, respectively. At the same time, blank control plates without any drugs were prepared. The mycelial cakes cut in step (3) were inoculated in the center of each drug-containing plate and blank control plate with the mycelial side facing down. Each concentration treatment and control group was repeated 3 times. The inoculated plates were placed in a constant temperature incubator at 26℃, protected from light and inverted for 5 days. After cultivation, take photos to record the mycelial growth (e.g. Figure 3As shown in the figure, the diameter of colonies on each plate was measured using the cross-cross method, the average value was calculated, and the mycelial growth inhibition rate was calculated according to formula (1). SPSS 27.0 software was used to perform statistical analysis on the experimental data, and the virulence regression equation and EC were calculated. 50 The values ​​are shown in Table 2.

[0079] (1).

[0080] Table 2. Virulence regression equation of *Actinidia kiwifruit* soft rot pathogen. Depend on Figure 3 As shown in Table 2, the EC of 2wt% cinnamaldehyde water emulsion is... 50 EC is 83.617 mg / L, 2 wt% carvacrol water emulsion. 50 EC of 36.961 mg / L, 2 wt% Litsea cubeba oil water emulsion 50 EC is 144.752 mg / L, 2 wt% thymol aqueous emulsion. 50 EC of 32.861 mg / L, 2 wt% thymol nanoliposomes 50 The concentration was 43.865 mg / L, indicating that thymol emulsion and thymol nanoliposomes have a good inhibitory effect on the pathogen of soft rot of kiwifruit.

[0081] Additionally, EC of 2wt% thymol nanoliposomes 50 The lower efficacy compared to the 2wt% thymol emulsion is due to the fact that in short-term (5-day) plate experiments, the thymol in the emulsion can immediately kill bacteria, resulting in strong acute antibacterial activity. In contrast, nanoliposomes require slow release, and the total amount of drug released within 5 days is relatively small, leading to a weaker measured immediate antibacterial activity. The advantage of liposomes lies in protecting the drug from degradation, providing longer-lasting protection through sustained release, and their nanoscale size allows for better penetration and targeting in vivo. Plate experiments cannot demonstrate these advantages. This data precisely proves the sustained-release properties of liposomes. To demonstrate "improved utilization," in vivo preservation experiments on kiwifruit are needed. Over a longer observation period, the actual efficacy and duration of action of liposomes are expected to be significantly better than those of the emulsion.

[0082] Test Example 3 Sustained-release performance test: The sustained-release performance of nanoliposomes was tested using the dialysis bag method. 2g of the thymol nanoliposome solution prepared in Example 1 was weighed and placed into a dialysis bag. After sealing, the bag was immersed in a beaker containing 500mL of 30wt% methanol solution. The bag was then stirred at 200rpm under constant temperature (25±2℃) conditions using a magnetic stirrer. 2mL of the supernatant was collected at predetermined intervals (2mL of 30wt% methanol was added after each sample) for liquid chromatography analysis. Each sample was repeated three times, and the average value was taken. Release curves were plotted, and commonly used zero-order kinetic models, first-order kinetic models, Higuchi release models, and Ritger-Peppas release models were selected for fitting analysis of the prepared release curves. The results are as follows: Figure 4 As shown in Table 3, Figure 4 Image A shows the sustained-release curve of thymol nanoliposomes. Figure 4 Table B shows the fitting analysis results of the release curve, and Table 3 shows the results of the fitting analysis with the release curve. Figure 4 Fitting results for different release models corresponding to B.

[0083] Table 3. Fitting results of different release models to the release curves Depend on Figure 4 As shown in Table 3, thymol nanoliposomes exhibited a significant burst release phenomenon in the initial stage of release from methanol, as shown in Figure A, with a cumulative release rate of 65.38% after 20 hours. Subsequently, the liposomes exhibited stable sustained-release characteristics. After 54 hours of release, the cumulative release rate reached 80.03%. This demonstrates that thymol nanoliposomes possess excellent sustained-release performance. The data on the release behavior of the nanoliposomes were fitted with different release models, such as... Figure 4 As shown in Table B and Table 3, combining the two, it can be seen that the release behavior of nanoliposomes fits the first-order kinetic model best, with R... 2 The value was 0.99473, followed by the Riter-Peppas release model and the Higuchi release model. 2 The values ​​were 0.9541 and 0.93943 respectively, with the zero-order dynamics model showing the worst fit. 2 The value was only 0.82382. The release behavior of the nanoliposomes showed the best fit to the first-order kinetic release model, conforming to the first-order kinetic equation and releasing according to the first-order release mechanism. Based on the fitting with the Riter-Peppas release model, the release characteristic index n of the liposomes was found to be 0.41, which is less than 0.45, indicating that its release mechanism is Fick diffusion.

[0084] Test Example 4 Kiwi fruit preservation effect test: (1) Comparison of the preservation effects of thymol nanoliposomes and carbendazim: To determine the preservation effect of thymol nanoliposomes on kiwifruit, a preservation evaluation experiment was conducted. Mature, unrotten kiwifruit fruits were selected for the evaluation. The commercially available fungicide carbendazim was used as a positive control, and sterile water was used as a blank control (CK). The specific test methods are as follows: 1) Fruit preparation and puncture: Use a sterile inoculation needle or pointed tweezers to make a uniform wound about 2-3 mm deep and about 1 mm in diameter in the middle of the kiwi fruit.

[0085] 2) Inoculation with pathogens: After the puncture wound, drip 10 μL of a pre-prepared suspension of spores of *Actinidia kiwifruit* (*Botrytis cinerea*) into each wound. 5 (spores / mL), air dry.

[0086] 3) Sample grouping and reagent preparation: The inoculated kiwifruit were divided into two large groups, and each large group was further divided into eight subgroups. The eight subgroups in each large group were treated with 0 mg / L (i.e., sterile water), 20 mg / L, 40 mg / L, 60 mg / L, 80 mg / L, 100 mg / L, 120 mg / L, and 140 mg / L of thymol nanoliposome aqueous solution (obtained by diluting the thymol nanoliposome solution prepared in Example 1 with sterile water, where 20 mg / L represents the mass of thymol provided by thymol nanoliposomes in 1L of diluted solution) and carbendazim aqueous solution (obtained by diluting commercially available carbendazim with sterile water, where 20 mg / L represents the mass of carbendazim in 1L of diluted solution). (The inoculated kiwifruit were immersed in the corresponding reagent for 2 minutes, and then removed and air-dried in a sterile and ventilated place). Each subgroup had three replicates.

[0087] 4) Storage and observation: Store the treated kiwifruit at 25℃ for 5 days. On the 5th day, take photos to record the extent of fruit rot (photos as shown). Figure 5 (as shown in the figure), and the diameter of the rotten area was measured. The average value of the results is shown in Table 4.

[0088] Table 4. Rot diameter of kiwifruit treated with different concentrations of carbendazim and thymol nanoliposomes Depend on Figure 5 As shown in Table 4, compared with the traditional pesticide carbendazim, the thymol nanoliposomes of the present invention can effectively inhibit the infection of kiwifruit soft rot pathogens, significantly reduce the incidence of kiwifruit disease, slow down the spread of lesions, and have a good inhibitory effect on kiwifruit soft rot.

[0089] (2) Comparison of the preservation effects of thymol nanoliposomes and thymol water emulsion: Using the aforementioned method, 100 mg / L thymol nanoliposome aqueous solution and 100 mg / L thymol water-emulsion solution (obtained by diluting the 2 wt% thymol water-emulsion from Test Example 2 with sterile water; 100 mg / L represents 100 mg of thymol provided by the water-emulsion in 1 L of diluted solution) were used as treatment agents, with sterile water serving as the blank control group (CK group). After treatment, the fruit was stored at 25℃ for 5 days, and the extent of fruit rot was recorded daily by photographing. On the 5th day, the kiwifruit skin was peeled open for observation. Results are as follows: Figure 6 As shown, on day 5, the lesion area in the CK group was significantly larger than that in both the 100 mg / L thymol nanoliposome aqueous solution and the 100 mg / L thymol aqueous emulsion solution, while the lesions in the 100 mg / L thymol nanoliposome aqueous solution were smaller than those in the 100 mg / L thymol aqueous emulsion solution. These results indicate that the 100 mg / L thymol nanoliposome aqueous solution has a stronger inhibitory effect on Staphylococcus aureus infection than the 100 mg / L thymol aqueous emulsion solution, and the actual preventive efficacy and duration of effect of the liposomes are significantly better than those of the aqueous emulsion.

[0090] 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 nanoliposomes, characterized in that, Includes the following steps: Thymol, cholesterol, and oleic acid are mixed to obtain an oil phase; polyethylene glycol, lauryl ether phosphate, emulsifying dispersant, and water are mixed to obtain an aqueous phase; the oil phase and the aqueous phase are mixed, emulsified and sheared at high speed, and then stirred at low speed to obtain the nanoliposomes.

2. The method for preparing nanoliposomes as described in claim 1, characterized in that, The mass ratio of thymol, cholesterol, and oleic acid is 2:2:

4.

3. The method for preparing nanoliposomes as described in claim 1, characterized in that, The mass ratio of polyethylene glycol, lauryl ether phosphate, emulsifying dispersant and water is 10:2:2:

78.

4. The method for preparing nanoliposomes as described in claim 1, characterized in that, The mass ratio of cholesterol in the oil phase to polyethylene glycol in the aqueous phase is 2:

10.

5. The method for preparing nanoliposomes as described in claim 1, characterized in that, The high-speed emulsification shearing is performed at a rotation speed of 6000-10000 rpm for a time of 3-10 min.

6. The method for preparing nanoliposomes as described in claim 1, characterized in that, The low-speed stirring is performed at a speed of 500-800 rpm for a duration of 0.5-2 hours.

7. Nanoliposomes prepared by the method of any one of claims 1-6.

8. The use of the nanoliposome as described in claim 7 in the preparation of an agent for preventing and treating soft rot in kiwifruit.

9. A preparation for controlling soft rot in kiwifruit, characterized in that, The raw materials include the nanoliposomes described in claim 7.

10. The use of a nanoliposome as described in claim 7 or an agent as described in claim 9 for preventing and controlling soft rot of kiwifruit in the prevention and control of soft rot of kiwifruit.

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