Ailanthus altissima extract nano-emulsion as well as preparation method and application thereof

Abstract

The invention discloses ailanthus altissima extract nano-emulsion and a preparation method and application thereof, the nano-emulsion comprises ailanthus altissima extract, holly essential oil, an emulsifier and water, and the ailanthus altissima extract is ailanthus altissima bark extract; the preparation method comprises the following steps: uniformly mixing the ailanthus altissima extract, the holly essential oil and the emulsifier as an oil phase, dropwise adding water into the oil phase while stirring, and continuously stirring after dropwise adding to obtain the uniform ailanthus altissima extract nano-emulsion. Compared with the ailanthus altissima extract, the ailanthus altissima nano-emulsion has the advantages that the insecticidal effect is obviously improved, the obvious insecticidal effect on various pests is achieved, the plant source pesticide is good in environmental compatibility, diversified in biological activity, safe to higher animals and natural enemies and not prone to generating drug resistance to pests.

Description

Technical Field

[0001] This invention relates to a pesticide and its preparation method, and particularly to an Ailanthus altissima extract nanoemulsion, its preparation method, and its application. Background Technology

[0002] With the advancement of agricultural modernization, traditional agricultural production methods urgently need transformation and upgrading to adapt to the demands of high-quality development. In the field of pest and disease control, exploring environmentally friendly and sustainable control strategies is particularly important. The extensive use of chemical pesticides has led to a series of environmental problems such as soil pollution and ecological imbalance. Finding new control methods is a crucial and effective way to solve these current problems. Plant-derived pesticides have complex active ingredients, diverse modes of action, and are less prone to resistance, making them an important direction for the creation of new pesticides.

[0003] The registered active ingredients in plant-derived pesticides in my country represent only a very small portion of the potential plant resources, and are mainly concentrated in a few mature varieties such as azadirachtin, matrine, osthol, and pyrethroids. To further expand the application of plant-derived pesticides, a systematic screening should be conducted focusing on my country's unique plant resources to provide sustainable solutions for green pest control in agriculture.

[0004] Ailanthus altissima (Mill.) Swingle, a deciduous tree belonging to the genus Ailanthus in the family Simaroubaceae, grows in northern, eastern, and southwestern my country, spanning 22 provinces from south to north. Its rapid growth, strong adaptability, and wide distribution provide abundant raw materials for the development of plant-derived pesticides. The leaves, bark, and fruits of Ailanthus altissima have a distinctive odor and insecticidal activity, commonly used in agricultural production for pest control, weeding, and disease prevention. However, the insecticidal efficacy of Ailanthus altissima extracts needs further improvement. Summary of the Invention

[0005] Objectives of the Invention: The first objective of this invention is to provide an ailanthus extract nanoemulsion that enhances the insecticidal effect of ailanthus extract; the second objective of this invention is to provide a method for preparing the ailanthus extract nanoemulsion; and the third objective of this invention is to provide applications of the ailanthus extract nanoemulsion.

[0006] Technical solution: The ailanthus extract nanoemulsion of the present invention includes ailanthus extract, wintergreen essential oil, emulsifier and water.

[0007] The wintergreen essential oil is used as a solvent.

[0008] Preferably, the ailanthus extract is an extract of the bark of the ailanthus tree. The bioactive compounds in the ailanthus bark extract include ailanthus ketone, ocotillone, and phellochin.

[0009] Preferably, the ailanthus bark extract is prepared by the following method: (1) pulverizing dried ailanthus bark, soaking it in ethanol, filtering it, collecting the filtrate, removing the solvent, and obtaining a crude extract; (2) dissolving the crude extract in water, extracting it with ethyl acetate, collecting the upper organic phase, removing the solvent, and obtaining the ailanthus bark extract.

[0010] Preferably, the emulsifier is EL-60.

[0011] Preferably, the nanoemulsion comprises the following components in the following weight fractions: 1%–5% ailanthus bark extract, 3%–15% holly essential oil, 6%–10% EL-60, and 70%–90% water.

[0012] Preferably, the droplet diameter of the nanoemulsion is 10-200 nm. If the particle size is too large, it will reduce the drug's targeting and hinder the effective release of the drug.

[0013] This invention screened holly essential oil as a solvent for ailanthus bark extract through extensive experiments, and EL-60 as an emulsifier, resulting in a stable nano-solution of ailanthus bark extract, which improved the insecticidal effect of ailanthus bark extract.

[0014] The preparation method of the ailanthus extract nanoemulsion of the present invention includes the following steps: mixing ailanthus extract, wintergreen essential oil and emulsifier as oil phase evenly, adding water dropwise to the oil phase while stirring, and continuing to stir after the addition is completed to obtain a uniform ailanthus extract nanoemulsion.

[0015] Preferably, the stirring is magnetic stirring, and the stirring speed is 600-1000 rpm.

[0016] The application of the Ailanthus altissima extract nanoemulsion described in this invention in the prevention or control of pests.

[0017] The pests mentioned are adult whiteflies, fall armyworm larvae, or Asian corn borer larvae.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The insecticidal effect of the ailanthus nanoemulsion of the present invention is significantly improved compared with the ailanthus extract; (2) Plant-derived pesticides have good environmental compatibility and diverse biological activities, are safe for higher animals and natural enemies, and are not prone to developing resistance to harmful organisms; (3) The preparation method of the ailanthus nanoemulsion of the present invention is simple. Attached Figure Description

[0019] Figure 1 The emulsifiers used in Examples 3 and 1 were freshly prepared using different emulsifiers. Figure 1 A) and after 14 days of heat storage at 54±2℃ ( Figure 1 B) Graph showing the changes in the appearance of the Ailanthus altissima extract nanoemulsion;

[0020] Figure 2 The centrifugal stability of the ailanthus extract nanoemulsions prepared with different emulsifiers for Example 3 and Comparative Example 1 after 7 days of cold storage at 0±2℃ is shown in the figure.

[0021] Figure 3 The graph shows the changes in particle size of the nanoemulsions of Ailanthus altissima extract prepared with different emulsifiers in Example 3 and Comparative Example 1, immediately after preparation, after 14 days of heat storage, and after 7 days of cold storage.

[0022] Figure 4 The following were prepared under different EL-60 weights for Examples 3 and 4 ( Figure 4 A) and after 14 days of heat storage at 54±2℃ ( Figure 4 B) Graph showing the changes in the appearance of the Ailanthus altissima extract nanoemulsion;

[0023] Figure 5 The centrifugal stability of the ailanthus extract nanoemulsions prepared under different EL-60 weights in Examples 3 and 4 after 7 days of cold storage at 0±2℃ is shown in the figure.

[0024] Figure 6 The graph shows the changes in particle size of the ailanthus extract nanoemulsions prepared under different EL-60 weights in Examples 3 and 4 immediately after preparation, after 14 days of heat storage, and after 7 days of cold storage.

[0025] Figure 7 The image shows the appearance of the Ailanthus altissima extract nanoemulsion prepared in Example 3 under a transmission electron microscope.

[0026] Figure 8 The surface tension diagram of the ailanthus extract nanoemulsion and water prepared in Example 3 is shown below.

[0027] Figure 9 They are respectively Ailanthus altissima extract nanoemulsion ( Figure 9 A) and 0.4% osthol soluble concentrate ( Figure 9 B) Contact angle diagram at the moment of initial contact with the front of the corn leaf;

[0028] Figure 10 The contact angle curves of the nanoemulsion prepared in Example 3 and the control group at different times on the front of a corn leaf;

[0029] Figure 11 The ailanthus extract nanoemulsions prepared in Example 3 are respectively ( Figure 11 A) and 0.4% osthol soluble concentrate ( Figure 11 B) The contact angle at the moment of initial contact with the back of the corn leaf;

[0030] Figure 12 The image shows the contact angle curves of the nanoemulsion prepared in Example 3 and the control group on the back of corn leaves at different times. Detailed Implementation

[0031] The technical solution of the present invention will be further described below with reference to the embodiments.

[0032] Example 1

[0033] The preparation of the extract from the bark of the tree of Ailanthus altissima includes the following steps:

[0034] (1) After the dried bark of the ailanthus tree is crushed into powder, it is soaked in industrial ethanol at room temperature (the mass ratio of ailanthus bark to ethanol is 1:10). After filtering the plant residue in the soaking liquid, the filtrate is collected. Under reduced pressure, the ethanol in the filtrate is evaporated to dryness using a rotary evaporator to obtain crude extract.

[0035] (2) Dissolve the obtained 435.2g crude extract in 1L of 25℃ warm water, extract five times with 1L of ethyl acetate, collect the upper organic phase, and evaporate the solvent under reduced pressure to obtain 187.9g of Ailanthus altissima extract sample.

[0036] Example 2

[0037] Screening of solvents for Ailanthus altissima extract: The bark extract of Ailanthus altissima was dissolved in fatty acid methyl ester, methyl oleate, S-100, S-150, S-200 and wintergreen essential oil, respectively. The solubility was tested in an ultrasonic cleaner at 25°C. The results are shown in Table 1.

[0038] Table 1. Solubility of Ailanthus altissima bark extract in different solvents

[0039]

[0040] As shown in Table 1, the bark of Ailanthus altissima has the best solubility in wintergreen essential oil, therefore wintergreen essential oil was chosen as the solvent.

[0041] Example 3

[0042] The nanoemulsion of ailanthus extract of the present invention comprises 4 wt% ailanthus bark extract, 12 wt% wintergreen essential oil, 9 wt% EL-60 and 75 wt% deionized water.

[0043] The method for preparing the ailanthus extract nanoemulsion includes the following steps: ailanthus bark extract, solvent, and emulsifier are mixed evenly in a beaker as the oil phase; an aqueous phase (deionized water) is added dropwise to the oil phase under magnetic stirring; after the addition is completed, stirring is continued at 1000 rpm for 20 minutes to obtain the ailanthus extract nanoemulsion.

[0044] Example 4

[0045] Based on Example 3, the amount of EL-60 was changed to 6wt%, 7wt%, 8wt%, and 10wt%, respectively, while the other conditions remained unchanged.

[0046] Example 5

[0047] The ailanthus extract nanoemulsion of the present invention comprises: 1% ailanthus bark extract, 3% holly essential oil, 6% EL-60, and 90% water.

[0048] Example 6

[0049] The ailanthus extract nanoemulsion of the present invention comprises: 5% ailanthus bark extract, 15% holly essential oil, 10% EL-60, and 70% water.

[0050] Comparative Example 1

[0051] Based on Example 1, EL-60 was changed to EL-10, EL-20, EL-40, and EL-80 respectively, while the other conditions remained unchanged.

[0052] Performance testing

[0053] 1. Emulsion stability test

[0054] The stability of the samples from Example 4 and Comparative Example 1 was tested using the following methods:

[0055] (1) The nanoemulsion sample was heat-stored at 54±2℃ for 14 days;

[0056] (2) Centrifugation of nanoemulsion samples after cold storage at 0±2℃ for 7 days;

[0057] (3) The particle size change of the nanoemulsion samples was tested using a Malvern laser particle size analyzer after 14 days of heat storage and 7 days of cold storage.

[0058] The test results are shown in the figure. Figure 1 and Figure 2 As shown.

[0059] like Figure 1 As shown, under different emulsifier preparation conditions ( Figure 1 A), except for EL-10, the other four surfactants can form nanoemulsions at room temperature; after heat storage at 54±2℃ for 14 days ( Figure 1 B) The appearance of the ailanthus extract nanoemulsion was uniform only when it was prepared with EL-60, while the nanoemulsions prepared with the other four surfactants showed floating oil or sediment at the bottom.

[0060] like Figure 2As shown, after cold storage at 0±2℃ for 7 days and centrifugation, the nanoemulsion prepared with surfactant EL-60 was the most stable, and the supernatant layer had the largest proportion in the nanoemulsion.

[0061] like Figure 3 As shown, when the emulsifier is EL-10, the particle size of the freshly prepared nanoemulsion is 207.5 nm, the particle size after 7 days of cold storage at 0±2℃ is 241.03 nm, and the particle size after 14 days of hot storage at 54±2℃ is 258.63 nm; when the emulsifier is EL-20, the particle size of the freshly prepared nanoemulsion is 147.37 nm, the particle size after 7 days of cold storage at 0±2℃ is 165.20 nm, and the particle size after 14 days of hot storage at 54±2℃ is 228.83 nm; when the emulsifier is EL-40, the particle size of the freshly prepared nanoemulsion is 189.73 nm, and the particle size after 7 days of cold storage at 0±2℃ is 258.63 nm. After 7 days of storage, the particle size was 240.97 nm; after 14 days of hot storage at 54±2℃, the particle size was 236.60 nm. When EL-60 was used as the emulsifier, the particle size of the freshly prepared nanoemulsion was 124.10 nm; after 7 days of cold storage at 0±2℃, the particle size was 138.80 nm; after 14 days of hot storage at 54±2℃, the particle size was 136.52 nm. When EL-80 was used as the emulsifier, the particle size of the freshly prepared nanoemulsion was 184.77 nm; after 7 days of cold storage at 0±2℃, the particle size was 276.60 nm; after 14 days of hot storage at 54±2℃, the particle size was 278.03 nm. In summary, EL-60 showed the best emulsification effect as an emulsifier.

[0062] 2. Stability and particle size testing of nanoemulsions under different emulsifier dosages

[0063] (1) The nanoemulsions obtained in Examples 3 and 4 were heat-stored at 54±2℃ for 14 days and their appearance was observed. The particle size was tested using a Malvern laser particle size analyzer.

[0064] (2) After storing the nanoemulsions obtained in Examples 3 and 4 at 0±2℃ for 7 days, the particle size was measured using a Malvern laser particle size analyzer, and the centrifugal stability was tested by centrifugation using a high-speed refrigerated centrifuge. The test results are as follows: Figures 4-6 As shown.

[0065] Depend on Figure 4 It can be seen that the newly prepared EL-60 under different weights ( Figure 4 A) and after 14 days of heat storage at 54±2℃ ( Figure 4 B) There are no obvious changes in appearance.

[0066] Depend on Figure 5It can be seen that, after cold storage at 0±2℃ for 7 days and centrifugation, the nanoemulsion prepared by surfactant EL-60 at a weight ratio of 9% was the most stable, and the supernatant layer had the largest proportion in the nanoemulsion.

[0067] Depend on Figure 6 The particle sizes of the nanoemulsions prepared with EL-60 dosages of 6wt%, 7wt%, 8wt%, 9wt%, and 10wt% were 132.4nm, 137.78nm, 123.33nm, 91.56nm, and 126.27nm, respectively. The particle sizes of the nanoemulsions after cold storage at 0±2℃ for 7 days were 156.37nm, 144.03nm, 142.17nm, 92.60nm, and 126.78nm, respectively. The particle sizes of the nanoemulsions after hot storage at 54±2℃ for 14 days were 133.73nm, 151.58nm, 143.24nm, 96.98nm, and 124.77nm, respectively. Among these, the particle size was smaller when the EL-60 dosage was 9wt%, and the particle size change was smaller after both cold and hot storage, indicating higher leaf surface adsorption and penetration efficiency.

[0068] The morphology of the freshly prepared nanoemulsion with 9wt% EL-60 is shown in the transmission electron microscope image. Figure 7 As shown.

[0069] Depend on Figure 7 It can be seen that the emulsion is uniformly dispersed, which is consistent with the results detected by the Malvern particle size analyzer.

[0070] 3. Surface tension test

[0071] (1) The surface tension of the nanoemulsion in Example 3 after dilution by 200 times was tested. The test results are as follows: Figure 8 As shown.

[0072] Depend on Figure 8 The surface tension of the nanoemulsion was found to be 33.875 mN / m, compared to 71.583 mN / m for deionized water. This indicates that the *Ailanthus altissima* extract nanoemulsion possesses good wettability, which is beneficial for improving application efficiency in practical applications.

[0073] 4. Plant surface contact angle test

[0074] The contact angles of the nanoemulsion (200 mg / L) prepared in Example 3, water, and 0.4% osthol soluble concentrate (200 mg / L) were tested at different times on the front and back of corn leaves. The test results are as follows: Figures 9-11 As shown.

[0075] Figure 9The contact angles of the nanoemulsion (9A) and the 0.4% osthol soluble concentrate (9B) upon initial contact with the front surface of corn leaves were 30.175° and 55.472°, respectively. Figure 10 It can be seen that the nanoemulsion of Ailanthus altissima extract has good wetting and spreading performance on the front side of corn leaves. In practical applications, it can improve the utilization rate of nanoemulsion and reduce its dosage.

[0076] Figure 11 The contact angles of the nanoemulsion (11A) and the 0.4% osthol soluble concentrate (11B) upon initial contact with the underside of corn leaves were 50.210° and 59.384°, respectively. Figure 12 It can be seen that the nanoemulsion of Ailanthus altissima extract has good wetting and spreading properties on the back of corn leaves.

[0077] 4. Insecticidal performance test of nanoemulsion

[0078] Test methods: The bioactivity test of adult whiteflies was conducted using a double-layer membrane method. The concentrations of the *Ailanthus altissima* extract nanoemulsion, *Ailanthus altissima* extract, and 0.4% osthol soluble concentrate prepared in Example 3 were 200 mg / L, 100 mg / L, 50 mg / L, 25 mg / L, 12.5 mg / L, 6.23 mg / L, and 3.13 mg / L, respectively. The bioactivity test of third-instar larvae of *Fall Armyworm* was conducted using a leaf-dipping method. The concentrations of the *Ailanthus altissima* extract nanoemulsion, *Ailanthus altissima* extract, and 0.4% osthol soluble concentrate prepared in Example 3 were... The concentrations were 800 mg / L, 400 mg / L, 200 mg / L, 100 mg / L, 50 mg / L, 25 mg / L, and 12.5 mg / L, respectively. The bioactivity test of the third instar larvae of the Asian corn borer was conducted using a feed-mixing method. The concentrations of the *Ailanthus altissima* extract nanoemulsion, *Ailanthus altissima* extract, and 0.4% osthol soluble concentrate prepared in Example 3 were 800 mg / L, 400 mg / L, 200 mg / L, 100 mg / L, 50 mg / L, 25 mg / L, and 12.5 mg / L, respectively. The test results are shown in Tables 2-4.

[0079] Table 2 Bioassay data of adult whiteflies

[0080]

[0081] As shown in Table 2, the effect of nanoemulsion is worse than that of commercial plant-derived pesticide 0.4% osthol soluble concentrate, but compared with unformulated ailanthus extract, the efficacy is increased by 3.37 times after 48 hours of treatment and by 1.86 times after 72 hours of treatment.

[0082] Bioassays were conducted using nanoemulsions of Ailanthus altissima extract from Examples 5 and 6 at a concentration of 50 mg / L, with adult whiteflies as the test insects.

[0083] Example 5: After treatment with Ailanthus altissima extract nanoemulsion for 48 hours, the mortality rate of whiteflies was 28%; after treatment for 72 hours, the mortality rate of whiteflies was 46%. Example 6: After treatment with Ailanthus altissima extract nanoemulsion for 48 hours, the mortality rate of whiteflies was 33%; after treatment for 72 hours, the mortality rate of whiteflies was 52%.

[0084] Table 3 Bioassay data of third instar larvae of the fall armyworm.

[0085]

[0086] As shown in Table 3, the efficacy of nanoemulsion increased by 1.24 times after 96 hours of treatment and by 1.66 times after 120 hours compared with untreated Ailanthus extract.

[0087] Table 4 Bioassay data of third-instar larvae of the Asian corn borer

[0088]

[0089] As shown in Table 4, the efficacy of nanoemulsion increased by 1.39 times after 96 hours of treatment and by 1.42 times after 120 hours compared with untreated Ailanthus extract.

[0090] 5. Safety assessment

[0091] (1) Safety assessment of Eisenia fetida

[0092] Test method: The nanoemulsion prepared in Example 3 was diluted to 320 mg / L, 160 mg / L, 80 mg / L, 40 mg / L, 20 mg / L, and 10 mg / L, respectively. Water was used as a control. The safety of Eisenia fetida was assessed by soil immersion method. The Eisenia fetida used in the experiment were raised for about three months and had obvious clitellum, with a weight of 300-600 mg / worm. The test results are shown in Table 5.

[0093] Table 5 Safety assessment of Eisenia fetidae

[0094]

[0095] Table 5 shows the LC-14 of the ailanthus extract nanoemulsion after 14 days of treatment. 50 The concentration is above 10 mg / kg (soil). According to the pesticide toxicity classification criteria for earthworms, the toxicity level to earthworms is low. The *Ailanthus altissima* extract nanoemulsion has low toxicity to non-target organisms in the environment and is relatively safe.

[0096] (2) Safety assessment of maize plants

[0097] Test method: A pot experiment was conducted in a greenhouse. The nanoemulsion prepared in Example 3 was diluted to 30 mg / L, 60 mg / L, 120 mg / L, and 240 mg / L, and sprayed on corn at the 5-6 leaf stage. A blank control group was set up. The growth of corn was investigated 14 days after spraying. The height from the ground surface to the corn growing point was taken as the plant height, and the plant height growth rate was calculated. The plant height growth rate can be calculated using the following formula:

[0098] Plant height growth rate = 14 × (plant height after treatment - plant height before treatment) / plant height before treatment

[0099] Table 6. Effects of Ailanthus altissima extract nanoemulsion on maize plant growth rate.

[0100]

[0101] Note: The same lowercase letter 'a' indicates no significant difference.

[0102] The effects of Ailanthus altissima extract nanoemulsion on the growth rate of maize plants are shown in Table 6. Compared with the blank control group, there was no significant difference in the growth rate of maize plants after 14 days of treatment with different concentrations of Ailanthus altissima extract nanoemulsion, and the maize plants did not show symptoms such as rotting, discoloration, or necrosis, indicating that the dosage used for treatment was insufficient to have an adverse effect on maize.

Claims

1. A nanoemulsion of Ailanthus altissima extract, characterized in that, It includes ailanthus extract, wintergreen essential oil, emulsifier, and water.

2. The ailanthus extract nanoemulsion according to claim 1, characterized in that, The ailanthus extract is an extract from the bark of the ailanthus tree.

3. The Ailanthus altissima extract nanoemulsion according to claim 1, characterized in that, The emulsifier is EL-60.

4. The Ailanthus altissima extract nanoemulsion according to claim 1, characterized in that, The nanoemulsion comprises the following components in the following weight fractions: 1%–5% Ailanthus altissima bark extract, 3%–15% Ilex chinensis essential oil, 6%–10% EL-60, and 70%–90% water.

5. The ailanthus extract nanoemulsion according to claim 4, characterized in that, The droplet diameter of the nanoemulsion is 10–200 nm.

6. The ailanthus extract nanoemulsion according to claim 2, characterized in that, The ailanthus bark extract is prepared by the following method: (1) the dried ailanthus bark is crushed, soaked in ethanol, filtered, the filtrate is collected, the solvent is removed, and a crude extract is obtained; (2) the crude extract is dissolved in water, extracted with ethyl acetate, the upper organic phase is collected, the solvent is removed, and the ailanthus bark extract is obtained.

7. A method for preparing the *Ailanthus altissima* extract nanoemulsion according to any one of claims 1 to 6, characterized in that, Includes the following steps: The ailanthus extract, wintergreen essential oil, and emulsifier were mixed evenly as the oil phase. Water was then added dropwise to the oil phase while stirring. After the addition was completed, stirring was continued to obtain a uniform ailanthus extract nanoemulsion.

8. The method for preparing the Ailanthus altissima extract nanoemulsion according to claim 7, characterized in that, The stirring is magnetic stirring, and the stirring speed is 600-1000 rpm.

9. The application of the ailanthus extract nanoemulsion according to any one of claims 1 to 6 in the prevention or control of pests.

10. The application according to claim 9, characterized in that, The pests mentioned are adult whiteflies, fall armyworm larvae, or Asian corn borer larvae.

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