A peppermint essential oil nanoemulsion, its preparation method and application

Abstract

The present invention provides a peppermint essential oil nanoemulsion, its preparation method and application, belonging to the technical field of plant pesticides; for the nanoemulsion, the raw materials include a surfactant, peppermint essential oil, and distilled water; first, castor oil polyoxyethylene ether and 1,2 - propanediol are mixed and stirred until a clear state is obtained as the surfactant; then the peppermint essential oil and the surfactant are mixed, and distilled water is added dropwise while continuing to stir, so that the solution changes from clear to viscous and turbid and then back to a clear and transparent state, and the peppermint essential oil nanoemulsion is prepared. The peppermint essential oil nanoemulsion can be used for the prevention and control of Panonychus ulmi. The present invention can effectively improve the stability and bioavailability of peppermint essential oil when used as a plant pesticide.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant pesticides, and particularly relates to a peppermint essential oil nanoemulsion, a preparation method thereof, and an application thereof. Background Art

[0002] At present, chemical pesticides such as bifenazate and cyflumetofen are mainly used to prevent mite pests in apple orchards. However, the long-term use of chemical pesticides not only makes plant pests and diseases resistant, but also causes serious harm to human health and environmental safety. To solve this problem, plant-derived pesticides have been developed. Plant-derived pesticides belong to a branch within the category of biological pesticides. It refers to plant-derived preparations that use the stable active ingredients contained in plants and, after being used on recipient plants by a certain method, enable the plants to avoid or reduce damage from harmful organisms such as diseases, insects, and weeds. Various plant-based pesticides are usually not a single compound, but all or part of the organic substances of the plant organism, with complex and variable compositions. Generally, they are included in various substances such as alkaloids, glycosides, toxic proteins, volatile essential oils, tannins, resins, organic acids, esters, ketones, and terpenes. Broadly speaking, plants rich in these highly physiologically active substances have the potential to be processed into pesticide preparations. Their quantity and type of substances are rich, and they are one of the sources of the third-generation pesticides that have attracted much attention at home and abroad. Using plant resources to control pests and diseases is a feasible way.

[0003] Peppermint ( Mentha haplocalyx Briq. ) is also called "Yindancao" and is a plant of the genus Mentha in the Lamiaceae family. Its leaves can be extracted to obtain peppermint essential oil. It has been reported that peppermint essential oil has rich and diverse functions, is pollution-free and residue-free in the environment. Peppermint essential oil is poorly soluble in water, volatile, and has low bioavailability. When using organic solvents to dissolve peppermint essential oil and using it in the field, it is found that it is easy to cause phytotoxicity to flowers and fruits and is easy to pollute the environment after use. The reason is that the formulation contains a large amount of organic solvents, such as benzene solvents like toluene, which have the risks of flammability, explosiveness, and easy poisoning due to their own chemical properties. The water emulsion of essential oil, which emulsifies peppermint essential oil in the form of oil-in-water, can solve the problems brought by organic solvents. However, in the preparation process of the water emulsion, in addition to adding emulsifiers, a large amount of thickeners, stabilizers, pH regulators and other substances are also required. Moreover, the water emulsion itself belongs to a thermodynamically unstable system. When peppermint essential oil is used as the oil phase, the phenomenon of droplet aggregation will occur, resulting in phenomena such as demulsification, delamination, and precipitation of the water emulsion, and ultimately making the product lose its commercial value and unable to conduct field efficacy tests. Summary of the Invention

[0004] The present invention overcomes the deficiencies of the prior art and provides a peppermint essential oil nanoemulsion, a preparation method thereof, and an application thereof, so as to improve the stability and bioavailability of peppermint essential oil when used as a plant pesticide.

[0005] To achieve the above object, the present invention is realized through the following technical solutions.

[0006] A nano-emulsion of peppermint essential oil, the raw materials include a surfactant accounting for 24.75% - 34.43% by mass percentage, peppermint essential oil accounting for 3.82% - 24.78%, and distilled water accounting for 50.47% - 61.75%.

[0007] Preferably, the raw materials consist of a surfactant accounting for 28.56% by mass percentage, peppermint essential oil accounting for 19.03%, and distilled water accounting for 52.41%.

[0008] Preferably, the surfactant is a mixture of castor oil polyoxyethylene ether and 1,2 - propanediol, and the mass ratio of castor oil polyoxyethylene ether to 1,2 - propanediol is 3:1 - 4:1.

[0009] More preferably, the mass ratio of castor oil polyoxyethylene ether to 1,2 - propanediol is 3:1.

[0010] A preparation method of a nano-emulsion of peppermint essential oil, comprising the following steps:

[0011] 1) Mix and stir castor oil polyoxyethylene ether and 1,2 - propanediol until it becomes clear, as the surfactant;

[0012] 2) Mix peppermint essential oil with the surfactant, and during the mixing process, add peppermint essential oil drop by drop to the surfactant, and stir while adding until it becomes uniform;

[0013] 3) Continue to stir and add distilled water drop by drop. When the solution changes from clear and transparent to viscous and turbid, continue to add distilled water drop by drop to the system while stirring. When the emulsion returns from turbid to clear and transparent, stop adding distilled water. At this time, the nano-emulsion of peppermint essential oil is prepared.

[0014] Preferably, the mixing and stirring in step 1) is carried out at room temperature.

[0015] More preferably, all the stirring involved in the steps is carried out using a magnetic stirrer.

[0016] A nano-emulsion of peppermint essential oil is used for the control of Panonychus ulmi.

[0017] A peppermint essential oil nano-emulsion with a mass percentage of 19.03% is diluted 40 times to control Panonychus ulmi.

[0018] The beneficial effects of the present invention compared with the prior art are:

[0019] The present invention prepares peppermint essential oil into a peppermint essential oil nanoemulsion preparation, characterizes its physicochemical properties, investigates and verifies the indoor toxicity and field efficacy of the peppermint essential oil nanoemulsion against Panonychus ulmi (Koch), and the 40-fold dilution of the 19.03% peppermint essential oil nanoemulsion has good field control efficacy against Panonychus ulmi (Koch), which is equivalent to that of the control group of 48% bifenazate suspension concentrate at 2000-fold dilution with no significant difference. The present invention provides a new idea for the development of peppermint essential oil as a botanical pesticide.

[0020] The active ingredient in the nanoemulsion has the smallest particle size, and the addition amount of the emulsifier during the preparation process is much higher than that of emulsifiable concentrates and aqueous emulsion concentrates. The oil-in-water nanoemulsion contains a large amount of water and has less phytotoxicity to plants. The peppermint essential oil nanoemulsion prepared in the present invention is a mixture composed of an aqueous phase, an oil phase, a surfactant, and a co-surfactant, and is a spontaneously formed transparent or semi-transparent microemulsion with particle sizes between 10 and 200 nm. It is a stable thermodynamic system, which can improve the transfer and absorption efficiency of macromolecular drugs inside plants, thereby improving the bioavailability of drugs inside plants, while improving the stability of drugs and reducing the volatility of drugs. The present invention uses nanoemulsion as the carrier of peppermint essential oil, and peppermint essential oil as the oil-phase drug. By adding a defined surfactant and co-surfactant to improve the emulsifying ability of the system, the optimal formulation of the peppermint essential oil nanoemulsion preparation for effectively controlling Panonychus ulmi (Koch) is prepared, and its physicochemical properties are characterized, and its stability is investigated, providing a scientific basis for the wide application of peppermint essential oil and nanoemulsion preparations. Description of the Drawings

[0021] Figure 1 It is the pseudo-ternary phase diagram for preparing peppermint essential oil nanoemulsion with 4 kinds of surfactants; among them, (1a) is Tween-80, (1b) is saponin, (1c) is EL-40, and (1d) is CO-40.

[0022] Figure 2 It is the appearance of the liquid after mixing different co-surfactants with EL-40; among them, (2a) is absolute ethanol, (2b) is polyethylene glycol, and (2c) is 1,2-propanediol.

[0023] Figure 3 It is the appearance diagram of the solution after mixing EL-40 and 1,2-propanediol in different mass ratios; among them, (3a) is the control, containing only EL-40, (3b) is 1:1, (3c) is 2:1, (3d) is 3:1, and (3e) is 4:1.

[0024] Figure 4 It is the pseudo-ternary phase diagram of the peppermint essential oil nanoemulsion.

[0025] Figure 5Tyndall effect phenomenon of peppermint essential oil nanoemulsion; among them, (5a) is the original solution of peppermint essential oil nanoemulsion, (5b) is diluted 10 times, (5c) is diluted 100 times, and (5d) is diluted 1000 times.

[0026] Figure 6 Morphology diagram of peppermint essential oil nanoemulsion particles observed under a transmission electron microscope.

[0027] Figure 7 Particle size distribution diagram of peppermint essential oil nanoemulsion.

[0028] Figure 8 Zeta potential distribution diagram of peppermint essential oil nanoemulsion. Detailed implementation manners

[0029] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer and more understandable, the present invention will be further described in detail in combination with embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. The technical solutions of the present invention will be described in detail below in combination with embodiments and drawings, but the protection scope is not limited by this.

[0030] Embodiment 1

[0031] Preparation of 3.82% peppermint essential oil nanoemulsion A, specifically the following steps:

[0032] 1. At room temperature, EL-40 and 1,2-propanediol are mixed and stirred in a mass ratio of 3:1 for 15 minutes. When the color is in a clear state, it is used as a surfactant.

[0033] 2. The peppermint essential oil and the surfactant are mixed in a mass ratio of 1:9. During the mixing process, the peppermint essential oil is added drop by drop to the surfactant, and stirred while adding until it is in a uniform state.

[0034] 3. Then continue to stir and add distilled water drop by drop. After observing that the solution suddenly changes from clear and transparent to viscous and turbid, continue to add distilled water drop by drop to the system while stirring, and observe that the emulsion changes from clear and transparent to turbid and then back to clear and transparent. At this time, the water content ratio is 61.75%.

[0035] Embodiment 2

[0036] Preparation of 11.93% peppermint essential oil nanoemulsion B, specifically the following steps:

[0037] 1. At room temperature, EL-40 and 1,2-propanediol are mixed and stirred in a mass ratio of 3:1 for 15 minutes. When the color is in a clear state, it is used as a surfactant.

[0038] 2. Mix peppermint essential oil and surfactant in a mass ratio of 3:7. During the mixing process, add the peppermint essential oil drop by drop to the surfactant, and stir while adding until a homogeneous state is achieved.

[0039] 3. Then continue stirring and add distilled water drop by drop. After observing that the solution suddenly changes from clear and transparent to viscous and turbid, continue to add distilled water drop by drop to the system while stirring, and observe that the emulsion changes from clear and transparent to turbid and then back to clear and transparent. At this time, the water content ratio is 52.41%.

[0040] Example 3

[0041] Preparation of 19.03% peppermint essential oil nanoemulsion C, specifically the following steps:

[0042] 1. At room temperature, mix EL-40 and 1,2-propanediol in a mass ratio of 3:1 and stir for 15 minutes. When the color is clear, use it as the surfactant.

[0043] 2. Mix peppermint essential oil and surfactant in a mass ratio of 4:6. During the mixing process, add the peppermint essential oil drop by drop to the surfactant, and stir while adding until a homogeneous state is achieved.

[0044] 3. Then continue stirring and add distilled water drop by drop. After observing that the solution suddenly changes from clear and transparent to viscous and turbid, continue to add distilled water drop by drop to the system while stirring, and observe that the emulsion changes from clear and transparent to turbid and then back to clear and transparent. At this time, the water content ratio is 52.41%.

[0045] Test Example 1

[0046] Screening of Surfactants

[0047] Screen four surfactants: Tween 80, tea saponin, EL-40, and CO-40. At room temperature, weigh peppermint essential oil and surfactant in mass ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1 respectively. Then add the peppermint essential oil drop by drop to the surfactant, and stir while adding until a homogeneous state is achieved. Then continue stirring and use a pipette to add distilled water drop by drop. Observe the state when the solution suddenly changes from clear and transparent to viscous and turbid, record the data, and continue to add distilled water drop by drop to the beaker until the liquid suddenly changes from viscous and turbid to clear and transparent again. Record the amount of distilled water added at the phase transition point and draw a pseudo-ternary phase diagram. Select the surfactant with the largest emulsification area as the screening result of the surfactant. See Figure 1 .

[0048] Test Example 2

[0049] Screening of Cosurfactants

[0050] Select anhydrous ethanol, polyethylene glycol (PEG-400), and 1,2-propanediol as co-surfactants for screening. Mix the best surfactant EL-40 screened in Test Example 1 with the three alternative co-surfactants at a ratio of 3:1, stir well, and place in a centrifuge tube. Centrifuge in a centrifuge at a speed of 3000 r / min for 15 minutes, observe the clarity, transparency, and layering of the liquid, and record the phenomena. Select the co-surfactant that is clear, transparent, and non-layered after mixing. See Table 1 and Figure 2 。

[0051]

[0052] Test Example 3

[0053] Determination of the ratio (Km) of surfactant to co-surfactant

[0054] Mix EL-40 and 1,2-propanediol at mass ratios of 1:1, 2:1, 3:1, and 4:1, stir well, place in a centrifuge tube, centrifuge in a centrifuge at a speed of 3000 r / min for 15 minutes, observe the changes in liquid layering or turbidity, compare the four, and compare with the control group with only EL-40. Select the best Km value with clarity, transparency, moderate viscosity, and no turbidity as the index. See Table 2 and Figure 3 。

[0055]

[0056] Test Example 4

[0057] Determination of the optimal formulation of nanoemulsion

[0058] According to the screening results of the above three experiments, mix peppermint essential oil with surfactants and co-surfactants, stir well, then use a magnetic stirrer to stir. While stirring, gradually add distilled water dropwise to the system, observe the phenomenon that the emulsion changes from clear and transparent to turbid and then back to clear and transparent, and record the amount of distilled water at the phase transition point. Calculate the mass fractions of each component of peppermint essential oil, mixed surfactant, and distilled water at the critical point of the system to determine the drug loading of peppermint essential oil nanoemulsion. Use the mass fractions of peppermint essential oil, distilled water, and mixed surfactant at the phase transition point as the three vertices, use Origin 2021 software to draw a pseudo-ternary phase diagram, and dilute the prepared peppermint essential oil nanoemulsion with distilled water to observe the stability after dilution, and determine the optimal ratio of the three phases of peppermint essential oil, mixed surfactant, and distilled water. See Table 3, Table 4, Figure 4 、Table 5.

[0059]

[0060]

[0061]

[0062] Test Example 5

[0063] Determination of the formation of nanoemulsions and identification of their structural types

[0064] When the peppermint essential oil nanoemulsion was irradiated with a laser pen, a bright path was formed in the liquid, namely the Tyndall effect. After the peppermint essential oil nanoemulsion was diluted 10 times, 100 times, and 1000 times, the Tyndall effect still existed. It was preliminarily judged that the peppermint essential oil nanoemulsion had been formed. The diffusion speed of the two dyes in the nanoemulsion was observed by dyeing method, and it was found that the diffusion speed of water-soluble methylene blue was significantly greater than that of Sudan red. Finally, it was judged that the type of the nanoemulsion was oil-in-water type (O / W). Figure 5 .

[0065] Test Example 6

[0066] Morphological observation and particle size, PDI and Zeta potential determination of peppermint essential oil nanoemulsion

[0067] The peppermint essential oil nanoemulsion prepared according to the parameters of each phase in the optimal formula was observed under a microscope. It was found that the peppermint essential oil nanoemulsion has obvious water-in-oil type (upper phase nanoemulsion) characteristics. The nanoemulsion coexists with excess water phase in the system. The water molecules in the outermost layer of each nanoemulsion particle fully wrap the oil molecules in the inner layer. The interfacial tension between the nanoemulsion and the water phase is much smaller than the interfacial tension between oil and water, forming a stable continuous phase system. The droplets are evenly distributed, uniform in shape and size, and spherical, but some particle droplets are elliptical, which is caused by the Ostwald ripening phenomenon. From the image, it can be judged that the peppermint essential oil nanoemulsion has good dispersibility. Figure 6 .

[0068] The particle size distribution of the prepared peppermint essential oil nanoemulsion was measured by Malvern laser particle size analyzer. It was found that the particle size distribution of the peppermint essential oil nanoemulsion was relatively uniform and generally normally distributed. The average particle size was 115.0 nm (n=3) and the polydispersity index PDI was 0.246. The instrument displayed the measurement result as: Result Meets Quality Criteria, that is, the particle size and polydispersity index of the prepared peppermint essential oil nanoemulsion met the product quality standards, see Figure 7 .

[0069] The measured Zeta potential of the peppermint essential oil nanoemulsion was -32.4 mV. The Zeta potential refers to the potential at the shear plane, also known as the electrokinetic potential or electrokinetic potential, which is an important indicator characterizing the stability of colloidal dispersions. The larger the absolute value of its value, the better the stability of the nanoemulsion. The instrument shows the measurement result as: Result Meets Quality Criteria, that is, the Zeta potential value of the prepared peppermint essential oil nanoemulsion meets the product quality standard, see Figure 8 .

[0070] Test Example 7

[0071] Indoor Bioassay

[0072] Indoor toxicity determination was carried out using the Potter spraying method. The peppermint essential oil nanoemulsion with a drug loading of 19.03% was successively diluted to 391, 781, 1563, 3125, 6250 mg / L. The apple tree leaves infested with Panonychus ulmi were placed under the Potter spraying tower for spraying treatment (pressure 100 KPa, volume 4 ml, sedimentation 2 min), then sealed and inverted, repeated 3 times, and the blank treatment was used as the control. Then it was placed in an incubator at a constant temperature of 26 °C for cultivation. The results were investigated at 12, 24, and 48 h respectively, and the number of dead Panonychus ulmi was recorded and the toxicity parameters of the peppermint essential oil nanoemulsion against Panonychus ulmi by spray contact were statistically analyzed. Those without response or unable to crawl normally after being touched with a writing brush were regarded as dead, and LC 50 was calculated, as shown in Table 6.

[0073] Mortality rate (%) = number of dead insects / total number of insects × 100

[0074] Corrected mortality rate (%) = 100 × (mortality rate of treatment group - mortality rate of control group) / (1 - mortality rate of control group).

[0075]

[0076] Test Example 8

[0077] Field Control Efficacy Determination

[0078] The field efficacy test was carried out in the orchard of Dongyang Village, Jinzhong City, Shanxi Province from June to July 2022, with an area of 1333.4 m 2(about 2 mu), with the variety of Red Fuji, tree age of 13 a, tree spacing of 3 m×4 m, and about 55 fruit trees planted per mu. The main pests include aphids, Panonychus ulmi, Carposina sasakii Matsumura, etc. The soil is loam, with a pH value of 8.3 and medium organic matter content. A total of 5 treatments were set up in the field experiment (see Table 7), and each treatment was repeated 4 times, arranged in a randomized block design. There were 2 trees in each plot, and a total of 20 plots were set up. On June 15th (the early and middle stages of mite damage), a manual sprayer was used to apply the medicine at a constant rate once, spraying the whole plant evenly. The amount of medicine applied was such that the front and back sides of the leaves were evenly covered with the medicine, and there was a slight dripping of the medicine droplets. There was no repeated spraying or missed spraying. The blank control was sprayed with an equal amount of water, and the water consumption per tree was 3.5 L. In order to theoretically achieve different degrees of control effects, the concentrations of 19.03% peppermint essential oil nanoemulsion in the field efficacy test were set as the LC 50 、LC 80 、LC 95 in the indoor 48-hour contact toxicity test. Then, the 19.03% peppermint essential oil nanoemulsion product needed to be diluted 304.33 times (diluted 300 times in actual application), 108.53 times (diluted 108 times in actual application), and 40.57 times (diluted 40 times in actual application) respectively. During the test period, no other pesticides were sprayed in all the test plots.

[0079]

[0080] The 40-fold solution of 19.03% peppermint essential oil nanoemulsion had a good field control effect on Panonychus ulmi, and there was no significant difference in the control effect compared with the 2000-fold solution of 48% bifenzate suspension concentrate in the control group.

[0081] The above content is a further detailed description of the present invention in combination with specific preferred embodiments. It cannot be determined that the specific embodiments of the present invention are limited to this. For those of ordinary skill in the technical field to which the present invention belongs, without departing from the premise of the present invention, several simple deductions or substitutions can still be made, and all should be regarded as belonging to the patent protection scope determined by the claims submitted for the present invention.

Claims

1. Application of a peppermint essential oil nanoemulsion, characterized in that, The peppermint essential oil nanoemulsion is used for controlling Panonychus ulmi; the raw materials of the peppermint essential oil nanoemulsion include a surfactant accounting for 24.75%-34.43% by mass percentage, peppermint essential oil accounting for 3.82%-24.78%, and distilled water accounting for 50.47%-61.75%; the surfactant is a mixture of castor oil polyoxyethylene ether and 1,2-propanediol, and the mass ratio of castor oil polyoxyethylene ether to 1,2-propanediol is 3:1-4:

1.

2. The application of a peppermint essential oil nanoemulsion according to claim 1, characterized in that, The peppermint essential oil nanoemulsion with a mass percentage of 19.03% is diluted 40 times to control Panonychus ulmi.

3. Use of a peppermint essential oil nanoemulsion according to claim 1, characterized in that, The raw materials of the peppermint essential oil nanoemulsion consist of a surfactant accounting for 28.56% by mass percentage, peppermint essential oil accounting for 19.03%, and distilled water accounting for 52.41%.

4. Use of a nanoemulsion of peppermint essential oil according to claim 1, characterized in that, The mass ratio of castor oil polyoxyethylene ether to 1,2-propanediol is 3:

1.

5. The application of a peppermint essential oil nanoemulsion according to claim 1, characterized in that, The preparation method of the peppermint essential oil nanoemulsion comprises the following steps: 1) Mix and stir castor oil polyoxyethylene ether and 1,2-propanediol until it becomes clear, as the surfactant; 2) Mix peppermint essential oil with the surfactant. During the mixing process, add peppermint essential oil drop by drop to the surfactant and stir continuously until it becomes homogeneous; 3) Continue stirring and add distilled water drop by drop. When the solution changes from clear and transparent to viscous and turbid, continue to add distilled water drop by drop to the system while stirring. When the emulsion returns from turbid to clear and transparent, stop adding distilled water. At this time, the peppermint essential oil nanoemulsion is prepared.

6. The application of a peppermint essential oil nanoemulsion according to claim 5, characterized in that, The mixing and stirring in step 1) are carried out at room temperature.

7. Use of the nanoemulsion of peppermint essential oil according to claim 5, characterized in that, All the stirring involved in the steps is carried out using a magnetic stirrer.

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