Long-acting cananga odorata essential oil nanoemulsion as well as preparation method and application thereof in killing mosquito larvae

Abstract

The invention discloses a long-acting cananga odorata essential oil nano-emulsion and a preparation method and application thereof in killing mosquito larvae, and the nano-emulsion comprises the following components in percentage by mass: 1-5% of cananga odorata essential oil, 2-4% of an emulsifier, 2% of polyvinylpyrrolidone (pvp) and the balance of deionized water. The preparation method comprises the following steps: (1) dissolving polyvinylpyrrolidone in deionized water to serve as a water phase; (2) mixing cananga odorata essential oil and an emulsifier, and carrying out ultrasonic treatment until the two are uniformly mixed to be transparent, so as to obtain an oil phase; and (3) adding the water phase into the oil phase under high-speed shearing of 10000-16000 rpm, and continuously shearing for 3-5 minutes to form the plant source mosquito larva killing nano emulsion. The compound has an obvious killing effect on larvae of aedes albopictus and can be used for biological control of aedes albopictus. The pesticide composition has high safety to non-target organisms, and pests do not easily generate drug resistance after the pesticide composition is used.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural application, and particularly relates to a long-acting plant-derived mosquito larvae killing nanoemulsion containing ylang-ylang essential oil, and a preparation method and application thereof. Background Art

[0002] Aedes albopictus is a container-breeding mosquito. At this stage, the larvae are relatively aggregated and easy to control in a centralized manner. Therefore, mosquito larvae killers have strong practical applications and control value. The current mainstream control method is mainly based on chemical control. However, some populations have developed resistance to it, and it is very likely to have an impact on non-target organisms and the environment. Compared with chemical pesticides, essential oils contain high concentrations of secondary metabolites, and because there are a large number of compounds with strong insecticidal activity against mosquitoes in plant essential oils, such as terpenes, monoterpenes and phenylpropanoids. In addition, essential oils are mixtures. Due to the synergistic effect of each component, they usually have stronger activity and are not easy to make mosquitoes resistant. And most plant essential oils are weakly toxic or even non-toxic to non-target organisms.

[0003] In order to reduce the problem of pesticide resistance caused by existing pesticide control, it is necessary to develop a new control method that can continuously control the larvae of Aedes albopictus.

[0004] In addition, since the larvae of Aedes albopictus mainly breed in various artificial and natural water containers in and around living rooms, the prepared preparations must have good water solubility to reduce the difficulty of application and will not cause harm to the applicators during the application process. They must be highly safe for non-target organisms and be able to interfere with the egg-laying and mating behavior of mosquitoes to reduce their reproduction rate, thereby achieving mosquito prevention and control effects in many aspects.

[0005] In order to achieve better control effects, the preparations used should be smaller in size so as to easily penetrate the cuticle of Aedes albopictus larvae and achieve better control effects. Summary of the invention

[0006] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0007] Based on the discovery that ylang-ylang essential oil has toxic activity against Aedes albopictus larvae, the object of the present invention is to provide a long-acting ylang-ylang essential oil mosquito larvicide prepared using ylang-ylang essential oil as a raw material, which does not contain any chemical pesticide components, has high toxicity to Aedes albopictus larvae, has high safety to non-target organisms, and is not easy for pests to develop drug resistance after use.

[0008] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0009] Therefore, the purpose of the present invention is to overcome the shortcomings of the prior art and provide a plant-based mosquito larvae killing nanoemulsion containing ylang-ylang essential oil, which comprises, by mass percentage, 1% to 5% of ylang-ylang essential oil, 2% to 4% of emulsifier, 2% of polyvinyl pyrrolidone (pvp), and the remainder is deionized water.

[0010] As a preferred embodiment of the plant-based mosquito larvae killing nanoemulsion containing ylang-ylang essential oil of the present invention, the emulsifier includes one or more of partial fatty acid esters of polyoxyethylene sorbitan, hydrogenated castor oil, and sorbitan fatty acid esters.

[0011] As a preferred embodiment of the plant-based mosquito larvae killing nanoemulsion containing ylang-ylang essential oil of the present invention, the polyoxyethylene sorbitan includes one or more of polysorbate-20, polyoxyethylene sorbitan monopalmitate, and polysorbate-80.

[0012] As a preferred embodiment of the plant-based mosquito larvae killing nanoemulsion containing ylang-ylang essential oil of the present invention, the hydrogenated castor oil includes one or more of castor oil polyoxyethylene ether (EL40) and castor oil polyoxyethylene ether-(80).

[0013] As a preferred embodiment of the plant-based mosquito larvae killing nanoemulsion containing ylang-ylang essential oil of the present invention, the sorbitan fatty acid ester includes one or more of sorbitan monolaurate, sorbitan monopalmitate, sorbitan monooleate, and sorbitan trioleate.

[0014] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing a plant-based mosquito larvae killing nanoemulsion containing ylang-ylang essential oil.

[0015] In order to solve the above technical problems, the present invention provides the following technical solutions, including:

[0016] (1) placing polyvinyl pyrrolidone in deionized water and completely dissolving the polyvinyl pyrrolidone in the water by magnetic stirring to form an aqueous phase; (2) mixing ylang-ylang essential oil and an emulsifier, ultrasonicating the mixture until the two are evenly mixed and transparent to obtain an oil phase; (3) adding the aqueous phase to the oil phase under high-speed shearing at 10,000 to 16,000 rpm, and shearing the resulting mixture for 3 to 5 minutes to form a plant-based mosquito larvae-killing nanoemulsion.

[0017] Another object of the present invention is to overcome the deficiencies in the prior art and provide a long-acting nanoemulsion containing ylang-ylang essential oil for use in killing mosquito larvae.

[0018] As a preferred application scheme of the plant-based mosquito larvae-killing nanoemulsion containing ylang-ylang essential oil of the present invention, the mosquito larvae include larvae of Aedes albopictus.

[0019] As a preferred application scheme of the plant-based mosquito larvae-killing nanoemulsion containing ylang-ylang essential oil of the present invention, the dosage of the aqueous emulsion is 25 mg / L, the required dosage of the agent is calculated by the volume of the water body according to the size of the mosquito larvae breeding site, and the aqueous emulsion is released into the water body in the form of spraying or natural dripping.

[0020] Beneficial effects of the present invention:

[0021] (1) The plant-based mosquito larvae-killing nanoemulsion containing ylang-ylang essential oil prepared by the high-energy emulsification method of the present invention has a significant killing effect on the larvae of Aedes albopictus and can be used for biological control of Aedes albopictus.

[0022] (2) Compared with chemical pesticides such as bispyrib, fenthion, and fenthion commonly used for Aedes albopictus, the botanical mosquito larvae killing nanoemulsion containing ylang-ylang essential oil provided by the present invention is a botanical pesticide. Ylang-ylang essential oil is a natural active substance, which leaves no residue after application and does not cause biological enrichment. It is easily degraded in the environment and is safe to mammals such as humans and livestock, natural enemies of pests and other beneficial organisms. It has the characteristics of low toxicity, high efficiency, safety and environmental protection. It does not have any residual components when used in field production and has no toxic effects on organisms other than target pests.

[0023] (3) The dosage form used in the present invention is mainly a nanoemulsion with strong water solubility, and no toxic emulsifier is used, which greatly reduces the damage to the environment and is safe for the applicator during the application process.

[0024] (4) The mechanism of action on mosquitoes is different from that of conventional pesticides, and mosquitoes are less likely to develop resistance.

[0025] (5) The raw material resources are abundant and low-cost, the preparation method is simple, and it is suitable for promotion and use.

[0026] (6) The present invention prepares ylang-ylang essential oil into a plant-derived nanoemulsion, which can greatly enhance the controlled release effect of the active ingredients of the essential oil, thereby prolonging the effect of killing mosquito larvae. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work. Among them:

[0028] Figure 1The figure shows the appearance changes of the nanoemulsion prepared in Example 1 of the present invention before and after storage, wherein the first on the left is the appearance of the just-prepared ylang-ylang essential oil nanoemulsion, the second on the left is the appearance of the ylang-ylang essential oil nanoemulsion after storage at 4°C for 14 days, and the third on the left is the appearance of the ylang-ylang essential oil nanoemulsion after storage at 54°C for 14 days.

[0029] Figure 2 The biological activity of ylang-ylang essential oil and its nanoemulsion at a concentration of 40 μg / mL against Aedes albopictus larvae after 24 hours. DETAILED DESCRIPTION

[0030] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0031] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0032] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0033] The raw materials used in the present invention are all commercially available unless otherwise specified.

[0034] In the embodiment of the present invention

[0035] 1. The test insects were Aedes albopictus, the strain was introduced from Jiangsu Provincial Center for Disease Control and Prevention, and the breeding conditions were 27°C ± 2°C, the relative humidity was 75 ± 5%, the photoperiod was 12h:12h, the larval stage was fed with complete nutrient mouse food, the adult stage was fed with 5% sucrose water, and the blood of mice was used for breeding.

[0036] 2. Test agent Ylang-ylang essential oil: purchased from Zhongxiang Natural Plant Co., Ltd., Ji'an City, Jiangxi Province.

[0037] The material prepared in the embodiment of the present invention is tested for performance using the following method, specifically:

[0038] 1. Measurement of nanoemulsion particle size: Malvern laser particle size analyzer (Zetasizer Nano

[0039] ZS90, UK) was used to measure the average droplet size of ylang-ylang essential oil nanoemulsion. The average droplet size of the nanoemulsion was measured. Before the measurement, the sample was diluted 200 times with deionized water to prevent the droplet density from affecting the measurement results. Three parallel experiments were performed at the same time.

[0040] 2. Mortality calculation formula: M = Nm / N × 100%

[0041] Where: M represents the mortality rate; Nm represents the number of dead insects; N represents the total number of test insects.

[0042] If the control mortality rate is less than 5%, no correction is required; if the control mortality rate is between 5% and 20%, correction is performed using the Abbott formula; if the control mortality rate is greater than 20%, the measurement is invalid.

[0043] Corrected mortality calculation formula: Mm = (Mt-Mc) / (1-Mc) × 100%

[0044] Where: Mm represents the adjusted mortality rate; Mt represents the mortality rate of the treatment group; Mc represents the mortality rate of the control group.

[0045] Example 1

[0046] This embodiment provides a method for preparing a long-acting ylang-ylang essential oil nanoemulsion, specifically:

[0047] Formula: 3% w / w ylang-ylang essential oil, 3.5% w / w emulsifier, 2% polyvinylpyrrolidone (pvp), and the balance is deionized water.

[0048] First, a certain amount of polyvinyl pyrrolidone is placed in deionized water, and the polyvinyl pyrrolidone is completely dissolved in the water by magnetic stirring to serve as the water phase. Then, ylang-ylang essential oil and emulsifier (EL40:EL80=1:2) are mixed and ultrasonicated for a period of time until the two are mixed and transparent, and then the ultrasonication is terminated to obtain the oil phase, and then the water phase is added to the oil phase under high-speed shearing of 10000 rpm, and the resulting mixture is sheared for 3.5 minutes to obtain the nanoemulsion.

[0049] The performance of the nanoemulsion prepared in this example was tested before and after storage, and the results are shown in Table 1 below.

[0050] Table 1

[0051] Day 0 Refrigerate at 4℃ for 14 days 54℃ heat storage for 14 days Particle size (nm) 101.2 101.7 100.9 Polydispersity Index PDI 0.204 0.2 0.211

[0052] Figure 1The graph of the appearance change of the nanoemulsion prepared in this example before and after storage shows that the newly prepared nanoemulsion exhibits blue light and is non-sticky without demulsification and precipitation. It also exhibits blue light and non-stickiness after hot storage and refrigeration for 14 days, which indicates that the prepared nanoemulsion is uniform and has good storage stability.

[0053] Example 2

[0054] The difference between this embodiment and embodiment 1 is that the content of ylang-ylang essential oil is adjusted to 1% w / w, and the rest of the preparation process is the same as that of embodiment 1 to prepare a nanoemulsion.

[0055] Example 3

[0056] The difference between this embodiment and embodiment 1 is that the content of ylang-ylang essential oil is adjusted to 5% w / w, and the rest of the preparation process is the same as that of embodiment 1 to prepare a nanoemulsion.

[0057] Comparative Example 1

[0058] The difference between this comparative example and Example 1 lies in the selection of essential oils. Essential oils are mainly screened for mosquito larvae killing activity and mosquito repellent activity to screen out essential oils with excellent activity in both aspects. The activity comparison results of different essential oils are shown in the following table:

[0059] Table 2: Difference in mortality of 3L larvae of Aedes albopictus at 100 mg / L concentration

[0060]

[0061]

[0062] Table 3: Repellent efficiency against mosquitoes at a concentration of 2 μL

[0063]

[0064] Comparative Example 2

[0065] The difference between this comparative example and Example 1 is that 2% w / w polyvinyl pyrrolidone was not added, and the rest of the preparation process was the same as that of Example 1 to obtain the corresponding emulsion.

[0066] Table 4:

[0067] Example 1 Comparative Example 2 Particle size (nm) 101.2 112.6 Polydispersity Index PDI 0.204 0.232

[0068] The nanoemulsion prepared in the above embodiment is tested for performance. We find that polyvinyl pyrrolidone can act as a solubilizing agent in the Ylang-ylang essential oil nanoemulsion system, increase the solubility of the poorly soluble Ylang-ylang essential oil, thereby reducing the particle size of the nanoemulsion and improving the solubility and bioavailability of the active ingredients in the nanoemulsion. And because polyvinyl pyrrolidone can form a thin film on the particle surface of the nanoemulsion, this film can be used as a barrier to slow down the diffusion rate of the active ingredients from the nanoemulsion to the external environment. In this way, polyvinyl pyrrolidone can prolong the release time of the active ingredients, thereby achieving a further sustained-release effect. Thereby finally achieving the long-lasting effect of killing larvae of the Ylang-ylang essential oil nanoemulsion.

[0069] Comparative Example 3

[0070] The difference between this comparative example and Example 1 is that the nanoemulsion is prepared by a low-energy emulsification method, and the rest of the preparation process is the same as that of Example 1 to obtain the corresponding emulsion.

[0071] Specifically, 3% (w / w) essential oil and 3.5% (w / w) emulsifier were mixed as the oil phase, 2% w / w polyvinyl pyrrolidone was dissolved in 91.5% (w / w) deionized water as the aqueous phase, and the aqueous phase was added dropwise to the oil phase and stirred at 500 rpm for 30 min to obtain an emulsion. The prepared emulsion could not present a uniform light blue state. The nanoemulsion prepared in the above embodiment was tested for performance, and the comparison results with those of Example 1 are shown in Table 2.

[0072] Table 5

[0073] Example 1 Example 2 Example 3 Particle size (nm) 101.2 150.1 151.2 Polydispersity Index PDI 0.204 0.379 0.228

[0074] As can be seen from the above table, adjusting the content of ylang-ylang essential oil has a significant effect on the performance of the nanoemulsion. The larger or smaller the essential oil content is, the smaller the particle size is. When the essential oil content is too low, the emulsifier and the essential oil are combined, and excess emulsifier will be produced, so that the excess emulsifier molecules are adsorbed on the droplet interface. When the emulsifier molecular chains are intertwined, the reduction in the droplet spacing may cause the droplets to merge. Resulting in an increase in particle size. If the essential oil content is too high, the emulsifier will not be able to completely wrap the droplets, but will instead cause the particle size to increase. According to the results in the above table, the best technical effect can be obtained when the content of ylang-ylang essential oil in the present invention is 3% w / w.

[0075] Example 4

[0076] The difference between this embodiment and embodiment 1 is that the content of the emulsifier is adjusted to 2% w / w, and the rest of the preparation process is the same as that of embodiment 1 to prepare a nanoemulsion.

[0077] Example 5

[0078] The difference between this embodiment and embodiment 1 is that the content of the emulsifier is adjusted to 2.25% w / w, and the rest of the preparation process is the same as that of embodiment 1 to prepare a nanoemulsion.

[0079] Example 6

[0080] The difference between this embodiment and embodiment 1 is that the content of the emulsifier is adjusted to 4% w / w, and the rest of the preparation process is the same as that of embodiment 1 to prepare a nanoemulsion.

[0081] Comparative Example 4

[0082] The difference between this comparative example and Example 1 is that the content of the emulsifier is adjusted to 0.5% (w / w), and the rest of the preparation process is the same as that of Example 1 to prepare a nanoemulsion.

[0083] The nanoemulsions prepared in the above examples and comparative examples were tested for performance, and the results compared with those of Example 1 are shown in Table 3.

[0084] Table 6

[0085] Example 1 Example 4 Example 5 Example 6 Comparative Example 4 Particle size (nm) 101.2 161.2 123.2 108.9 164.3 Polydispersity Index PDI 0.204 0.234 0.213 0.209 0.241

[0086] As can be seen from the above table, adjusting the content of the emulsifier has a significant effect on the particle size of the nanoemulsion, because the emulsifier reduces the interfacial tension of the two phases, thereby reducing the free energy of the nanoemulsion formation and reducing the particle size of the droplets. Too low an emulsifier content will form a thicker multilayer adsorption film, a dense interfacial film and produce a lower interfacial tension, which will have an adverse effect on the nanoemulsion. According to the results in the above table, the best technical effect can be obtained when the content of the emulsifier in the present invention is 3.5% w / w.

[0087] Example 7

[0088] The difference between this embodiment and embodiment 1 is that the shearing time is adjusted to 1 min, and the rest of the preparation process is the same as that of embodiment 1 to prepare a nanoemulsion.

[0089] Example 8

[0090] The difference between this embodiment and embodiment 1 is that the shearing time is adjusted to 3 minutes, and the rest of the preparation process is the same as that of embodiment 1 to prepare a nanoemulsion.

[0091] Example 9

[0092] The difference between this embodiment and embodiment 1 is that the shearing time is adjusted to 5 minutes, and the rest of the preparation process is the same as that of embodiment 1 to prepare a nanoemulsion.

[0093] The performance of the nanoemulsion prepared in the above example was tested, and the results compared with those of Example 1 are shown in Table 4.

[0094] Table 7

[0095] Example 1 Example 7 Example 8 Example 9 Particle size (nm) 101.2 171 124.4 109.9 Polydispersity Index PDI 0.204 0.24 0.206 0.241

[0096] As can be seen from the table above, the effect of adjusting the shearing time on the performance of the nanoemulsion is significant. If the shearing time is too short, all the droplets cannot be broken into tiny droplets, and if the shearing time is too short, the probability of collision between the droplets is reduced, so that the two combinations are uneven, resulting in larger particles. If the shearing time is too long, a large amount of energy input will be generated, thereby causing the system to heat up significantly, affecting the stability of the emulsion itself and even causing demulsification. According to the results in the table above, the best technical effect can be obtained when the shearing time in the present invention is 3.5min.

[0097] Example 10

[0098] The difference between this embodiment and embodiment 1 is that the types of emulsifiers are adjusted to castor oil polyoxyethylene ether EL-10, castor oil polyoxyethylene ether EL-20, castor oil polyoxyethylene ether EL-40, castor oil polyoxyethylene ether EL-60, castor oil polyoxyethylene ether EL-80, and castor oil polyoxyethylene ether EL-90, and the rest of the preparation process is the same as that of embodiment 1 to obtain a nanoemulsion.

[0099] The results showed that after 7 days of hot storage, the appearance of the ylang-ylang essential oil nanoemulsions prepared with different emulsifiers was different. Among them, castor oil polyoxyethylene ether EL-10, castor oil polyoxyethylene ether EL-20 ether EL-60, and castor oil polyoxyethylene ether EL-90 showed oil-water interface stratification, and the nanoemulsions prepared on the surface could not maintain a stable system. In contrast, castor oil polyoxyethylene ether EL-40 and castor oil polyoxyethylene ether EL-80 showed blue light and were non-sticky without demulsification and precipitation. The nanoemulsions prepared on the surface were relatively uniform and stable.

[0100] Example 11 Biological activity of ylang-ylang essential oil and its nanoemulsion against 3L larvae of Aedes albopictus after 24 hours of treatment

[0101] The activity of the mosquito larvae-killing nanoemulsion against the third-instar larvae of Aedes albopictus was determined by the larval immersion method:

[0102] Based on the preliminary experiment, the third-instar larvae were placed in glassware containing 100 ml of 25 μg / ml, 35.00 μg / ml, 45.00 μg / ml, 55.00 μg / ml, and 65.00 μg / ml respectively, in which the ylang-ylang essential oil was dissolved with a certain amount (1 mL) of ethanol and evenly dispersed in the 100 mL glassware. A blank control group containing ethanol was also set up.

[0103] The nanoemulsion prepared in Example 1 was diluted with deionized water to different concentration gradients: 8.00 μg / ml, 16.00 μg / ml, 24.00 μg / ml, 32.00 μg / ml, and 40.00 μg / ml. A blank nanoemulsion (without ylang-ylang essential oil) was also prepared, with the concentration set to 40.00 μg / ml.

[0104] In a greenhouse at 25℃±1℃ and relative humidity of 60% to 80%, treat 3rd instar Aedes albopictus larvae with the above dilution, and check the number of deaths after 24 hours. Each treatment has 20 larvae, and the process is repeated 5 times.

[0105] The organic solvent itself was used as a control. According to the mortality rate corresponding to each concentration, the dose was taken logarithm, the mortality rate was converted into probability units for regression, and the LC 50 and LC 90 value.

[0106] The following table shows the results of the biological activity test of ylang-ylang essential oil and its nanoemulsion on 3L larvae of Aedes albopictus after 24 hours of treatment.

[0107] Table 8 Bioactivity of ylang-ylang essential oil and its nanoemulsions against 3L larvae of Aedes albopictus after 24h treatment

[0108]

[0109] As can be seen from the table above, by using different concentrations of ylang ylang essential oil and ylang ylang essential oil nanoemulsion to test the toxicity of 3L larvae of Aedes albopictus, it can be seen that ylang ylang essential oil itself has better larvicidal activity. 50 The LC value of Ylang-ylang essential oil nanoemulsion was 48.44 μg / ml (46.12 μg / ml-50.70 μg / ml), which was better than some plant essential oils. Secondly, we found that the larvicidal activity of Ylang-ylang essential oil nanoemulsion was better than that of Ylang-ylang essential oil itself. 50 The concentration of nanoemulsion was 22.63 μg / ml (20.64 μg / ml-24.45 μg / ml), which may be because the nanoemulsion particles are smaller and can more easily penetrate the epidermis of the larvae of Aedes albopictus, thus having a better synergistic effect, with a synergistic multiple of about 2.14 times. This shows the potential of ylang-ylang essential oil nanoemulsion as a new type of green larval control agent.

[0110] Figure 2The biological activity of ylang-ylang essential oil and its nanoemulsion on Aedes albopictus larvae at a concentration of 40 μg / mL after 24 hours. It can be seen that the same concentration of ylang-ylang essential oil and mosquito-killing nanoemulsion are treated for 24 hours, and the lethality of ylang-ylang essential oil nanoemulsion is increased by nearly 60% compared with ylang-ylang essential oil, indicating that the mosquito-killing nanoemulsion prepared by the present invention has good biological activity. The long-term effect is reflected in that the ylang-ylang essential oil nanoemulsion can maintain strong biological activity within 3 days compared with the unstable and volatile characteristics of ylang-ylang essential oil itself.

[0111] Safety of Ylang Ylang essential oil nanoemulsion to non-target organisms:

[0112] ① Acute toxicity to zebrafish: The static experimental method in GB / T 31270 was used to evaluate the acute toxicity of nanoemulsions to zebrafish.

[0113] Different doses of Ylang Ylang essential oil nanoemulsion were added to a glass vessel containing 1000 mL of water and mixed thoroughly to prepare five different concentrations of nanoemulsions in aqueous solution (8.00 μg / ml, 16.00 μg / ml, 24.00 μg / ml, 32.00 μg / ml, 40.00 μg / ml). Ten zebrafish of similar size and weight were selected and placed in a glass vessel with 3 replicates per group. The experimental temperature was maintained at 23 °C ± 2 °C, and the light-dark ratio was 12:12. The mortality of zebrafish was recorded, and the LC was calculated at 24, 48, 72, and 96 h. 50 The evaluation criteria for the classification of pesticide toxicity to zebrafish refer to the "Chemical Pesticide Environmental Safety Evaluation Test Standards", and the test results are shown in the following table.

[0114] Table 9 Toxicity determination of nanoemulsion to zebrafish

[0115] Processing time (h) <![CDATA[LC 50 (mg / kg)]]> 95% Confidence Interval Regression independence curve Chi(Sig) toxicity 24 33.63 31.73-35.19 y=10.89x-16.64 2.68 Low 48 32.90 30.84-34.48 y=11.17x-16.94 1.90 Low 72 32.23 29.90-33.87 y=10.83x-16.32 2.29 Low 96 31.51 28.99-33.19 y=10.97x-16.42 1.41 Low

[0116] Table 7 shows the acute toxicity of ylang ylang essential oil nanoemulsion to zebrafish. The acute toxicity of ylang ylang essential oil nanoemulsion to zebrafish within four days is as follows: LC values ​​for zebrafish at 24h, 48h, 72h, and 96h 50 The LC values ​​of Ylang-ylang essential oil nanoemulsion on zebrafish in different time periods were 33.63 mg / kg, 32.90 mg / kg, 32.23 mg / kg and 31.51 mg / kg respectively. 50 Both were significantly higher than 10 mg / kg, indicating that ylang-ylang essential oil nanoemulsion has relatively good safety for zebrafish.

[0117] ②Ylang-ylang essential oil nanoemulsion's proliferative activity on human liver cells:

[0118] In order to verify the effect of Ylang Ylang essential oil nanoemulsion on the human body, L02 cells were selected to investigate its potential cytotoxicity. L02 cells were placed in RPMI 1640 complete medium (10% fetal bovine serum, 1% streptomycin) and cultured at 37°C and 5% CO2 saturated humidity. The cells used were in the logarithmic growth phase (fusion rate ≥ 80%). The cytotoxicity of cypermethrin nanoemulsion was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. L02 cells were cultured in 96-well plates and the maximum culture medium (100 μL) was added. Then, the same dose of (50 μg / mL, 100 μg / mL) nanoemulsion was added to each well. After 24 and 48 h of culture, CCK-8 (10 μL) was added to the wells under light-proof conditions. After incubation for 2 h, the absorbance at 490 nm was measured using a microplate reader (SPARK, Tecan, Switzerland). The relative survival rates of cells after incubation for 24 h and 48 h under different concentrations of ylang-ylang essential oil nanoemulsion are shown in Table 8 below.

[0119] Table 10

[0120] 24h relative cell survival rate (%) 48h relative cell survival rate (%) 50 μg / mL 95.7％ 87.9％ 100 μg / mL 91.8％ 83.1％

[0121] In this experiment, we used CCK8 to detect the proliferation activity of human hepatocytes after 24h and 48h of incubation, and used the treatment of nanoemulsion without adding ylang ylang essential oil as a control to calculate the relative cytotoxicity of different concentrations of ylang ylang essential oil nanoemulsion to human hepatocytes (L02). The experimental results are shown in the table above: relative to the 100μg / mL treatment group, the overall cell survival rate of 50μg / mL ylang ylang essential oil nanoemulsion was higher than 100μg / mL whether it was incubated for 24h or 48h. We also found that in cells treated with ylang ylang essential oil nanoemulsion, the overall cell viability was at a higher level, indicating that ylang ylang essential oil nanoemulsion has low toxicity to human hepatocytes.

[0122] In summary, the plant-based mosquito larvae-killing nanoemulsion containing ylang-ylang essential oil prepared by the high-energy emulsification method of the present invention has a significant killing effect on all insect stages of Aedes albopictus, and can be used for comprehensive biological control of Aedes albopictus. Compared with conventional pesticides, the mechanism of killing mosquito larvae by plant essential oils is relatively complicated, mainly because the composition of essential oils is complex because essential oils are a mixture of various compounds, and different components may have different modes of action, that is, the possible targets are diverse and not easy to cause the improvement of mosquito resistance, and there may be synergistic effects between the various modes of action. At present, the main targets of essential oils for killing mosquito larvae may be the following: causing protein denaturation of mosquito larvae, inhibition of related enzymes, and destruction of membrane systems. In addition, plant-based substances still have good insecticidal activity for certain strains that cause resistance, and mosquitoes are not easy to develop resistance.

[0123] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A long-acting ylang-ylang essential oil nanoemulsion, characterized in that: Calculated by mass percentage, it includes 1% to 5% of ylang-ylang essential oil, 2% to 4% of emulsifier, 2% of polyvinyl pyrrolidone, and the balance is deionized water.

2. The long-acting ylang-ylang essential oil nanoemulsion according to claim 1, characterized in that: The emulsifier includes one or more of partial fatty acid esters of polyoxyethylene sorbitan, hydrogenated castor oil, and sorbitan fatty acid esters.

3. The long-acting ylang-ylang essential oil nanoemulsion according to claim 2, characterized in that: The polyoxyethylene sorbitan includes one or more of polysorbate-20, polyoxyethylene sorbitan monopalmitate, and polysorbate-80.

4. The long-acting ylang-ylang essential oil nanoemulsion according to claim 2, characterized in that: The hydrogenated castor oil includes one or more of castor oil polyoxyethylene ether (EL40) and castor oil polyoxyethylene ether-(80).

5. The long-acting ylang-ylang essential oil nanoemulsion according to claim 2, characterized in that: The sorbitan fatty acid ester includes one or more of sorbitan monolaurate, sorbitan monopalmitate, sorbitan monooleate, and sorbitan trioleate.

6. The method for preparing the long-acting ylang-ylang essential oil nanoemulsion according to any one of claims 1 to 5, characterized in that: The following steps are involved: (1) polyvinyl pyrrolidone is placed in deionized water and stirred to completely dissolve the polyvinyl pyrrolidone in the water to form an aqueous phase; (2) ylang-ylang essential oil and an emulsifier are mixed and ultrasonicated until the mixture becomes transparent to obtain an oil phase; (3) the aqueous phase is added to the oil phase under high-speed shearing at 10,000 to 16,000 rpm, and the resulting mixture is sheared for 3 to 5 minutes to form a plant-based mosquito larvae-killing nanoemulsion.

7. Use of the long-acting ylang-ylang essential oil nanoemulsion obtained according to any one of claims 1 to 5 for killing mosquito larvae.

8. The use according to claim 7, characterized in that: The mosquito larvae include Aedes albopictus larvae.

9. The use according to claim 7, characterized in that: The method of use is: according to the dosage of 20~30mg / L of water emulsion, according to the size of the mosquito larvae breeding site, the required amount of medicine is calculated through the water volume, and the water emulsion is released into the water body by spraying or natural dripping.

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