Litsea cubeba nano-emulsion as well as preparation method and application thereof

Abstract

The invention firstly discloses a preparation method of a litsea cubeba nano-emulsion, which comprises the following steps: soaking litsea cubeba in methanol in a dark place for 72 hours, filtering, and carrying out reduced pressure rotary evaporation on filtrate to obtain a methanol crude extract; and carrying out liquid-liquid extraction on the methanol crude extract, and continuously carrying out reduced pressure rotary evaporation to obtain the litsea cubeba petroleum ether extract. The method comprises the following steps: sucking 1mL of an emulsifier EL-80, then sucking 1.5 mL of a solvent S-200, and uniformly mixing the emulsifier EL-80 and the solvent S-200. Then, weighing 1g of litsea cubeba extract, dissolving the litsea cubeba extract in the mixed solvent, and stirring at room temperature at the speed of 1000rpm for 15 minutes, so that the litsea cubeba extract and the mixed solvent are uniformly mixed to obtain an oil phase; and the water phase is deionized water. Under magnetic stirring, dropwise adding the oil phase into the water phase, and continuously stirring for 20 minutes at the speed of 1000rpm after dropwise adding is finished, so as to obtain the litsea cubeba nano-emulsion. According to the litsea cubeba nano-emulsion prepared by the invention, not only can the dosage of the litsea cubeba extract during insect killing be reduced, but also the insecticidal effect of the litsea cubeba extract is enhanced, and a new thought is provided for research and development and industrial production of litsea cubeba insecticides.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical industry, and particularly relates to a method for preparing a litsea cubeba nanoemulsion and the litsea cubeba nanoemulsion prepared by the preparation method, and also relates to application of the litsea cubeba nanoemulsion in preparing a botanical insecticide for preventing and controlling oriental armyworm. Background Art

[0002] The Oriental armyworm is a major migratory pest on my country's three major staple crops, corn, wheat and rice. It was listed in the national list of Class I crop pests and diseases in 2020, and presents seasonal north-south migration damage in large areas of my country every year. There are about 8-2 generations from south to north in my country each year, mainly harming the late rice generation in autumn and the wheat generation in winter. When the Oriental armyworm is 1-2 years old, it bites the leaf flesh, causing transparent stripes on the leaves. After the 3rd instar, it eats wheat leaves along the leaf margins to form notches. As a typical voracious pest, in severe cases, it will eat the wheat stalks bare, and it can bite off the ears or bite the twigs and stalks during the ear stage, causing a large amount of grain falling. In severe cases, the crop can be eaten up within 1-2 days, causing serious grain production losses.

[0003] At present, the main control methods include agricultural control and chemical control. For example, ducks can be released to peck at the larvae of the Oriental Armyworm during the outbreak period. Both chemical insecticides and Bt have good control effects on the Oriental Armyworm. However, due to the large-scale and unreasonable and frequent use of drugs, the Oriental Armyworm has developed resistance to most insecticides, resulting in an exponential increase in the amount of drugs used, which in turn has caused serious damage to non-target organisms and the natural environment.

[0004] Litsea cubeba belongs to the genus Litsea cubeba of the Lauraceae family. It is a perennial deciduous shrub or small tree plant, mostly used as a traditional Chinese medicine. However, various extracts of Litsea cubeba, including essential oils, have different degrees of repelling, killing and inhibiting cell growth, development and reproduction on pests. During the applicant's experiment, it was found that Litsea cubeba extract has good toxic activity (stomach poison and contact toxicity) against Oriental armyworm. As a plant-derived extract, Litsea cubeba extract contains a variety of compounds, and Oriental armyworm is not easy to develop resistance to it. Compared with chemical synthetic pesticides, the residue in the natural environment is low. As a botanical insecticide for the prevention and control of Oriental armyworm, it is an ideal choice to replace chemical synthetic insecticides that have already developed resistance.

[0005] Therefore, it is of great practical significance to study and develop a litsea cubeba nanoemulsion. Summary of the invention

[0006] In order to solve the above technical problems, an insecticidal nanoemulsion system, preparation method, application and application method are provided. The technical scheme uses nanoemulsification technology to make the extract of Litsea cubeba into a nanoemulsion system, which not only reduces the dosage of the extract of Litsea cubeba, but also has excellent stability and good insecticidal effect.

[0007] In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0008] The present invention first discloses a method for preparing a litsea cubeba nanoemulsion, comprising:

[0009] (1) Soaking the seeds of Litsea cubeba in methanol in the dark for 72 hours, filtering, and taking the filtrate and performing vacuum rotary evaporation to obtain a crude methanol extract for later use;

[0010] (2) liquid-liquid extraction of the crude methanol extract, continued vacuum rotary evaporation, and air-dried to obtain a litsea cubeba petroleum ether extract;

[0011] (3) 1 mL of emulsifier EL-80 and 1.5 mL of S-200 aromatic solvent were taken, and the two were mixed evenly. Then, 1 g of petroleum ether extract of Litsea cubeba was weighed and added thereto, and stirred evenly at room temperature to obtain an oil phase for later use;

[0012] (4) adding the oil phase drop by drop into the water phase while stirring, and continuing to stir after the addition is completed, thereby obtaining a litsea cubeba nanoemulsion.

[0013] Furthermore, the stirring conditions at room temperature in step (3) are: stirring speed is 1000 rpm, and stirring time is 15 min.

[0014] Furthermore, the aqueous phase in step (4) is deionized water.

[0015] Furthermore, the amount of deionized water used is 97 mL.

[0016] Furthermore, the conditions for continuous stirring in step (4) are: stirring speed is 1000 rpm, and stirring time is 20 min.

[0017] The invention also discloses a litsea cubeba nanoemulsion prepared by any of the above preparation methods.

[0018] The invention also discloses an application of the litsea cubeba nanoemulsion in preparing a botanical insecticide for preventing and controlling oriental armyworm.

[0019] Furthermore, in the botanical insecticide, the concentration of the Litsea cubeba nanoemulsion is 10 mg / mL, 5 mg / mL, and 2.5 mg / mL.

[0020] The beneficial effects of the present invention are:

[0021] The droplet particle size of the litsea cubeba nanoemulsion formed by the present invention is small (<180nm), the nanoemulsion system is stable at (0±2)°C, and the particle size and Zeta potential change range is small. After being stored for 7 days, the nanoemulsion is centrifuged without stratification, and has strong stability. At the same time, the amount of emulsifier and solvent used is small, which improves environmental safety.

[0022] The invention prepares the Litsea cubeba extract into a nanoemulsion through nanoemulsification technology, thereby enhancing the insecticidal effect of the Litsea cubeba extract at the same concentration, and provides a new idea for the research and development and industrial production of Litsea cubeba insecticides.

[0023] The raw materials used in the preparation of the litsea cubeba nanoemulsion formed by the invention are simple and easily available, the reaction conditions are mild, and the operation is simple and convenient. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the daily average particle size change of Litsea cubeba nanoemulsion at (0±2)℃;

[0025] Figure 2 is the daily Zeta potential change of Litsea cubeba nanoemulsion at (0±2)℃;

[0026] Figure 3 This is a microstructure analysis diagram of the Litsea cubeba nanoemulsion prepared in the present invention;

[0027] Figure 4 The centrifugal stability of Litsea cubeba nanoemulsion stored at (0±2)°C for 7 days;

[0028] Figure 5 The toxicity of Litsea cubeba extract and Litsea cubeba nanoemulsion against the oriental armyworm. DETAILED DESCRIPTION

[0029] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are only examples, and those skilled in the art may think of other obvious variations.

[0030] Unless otherwise specified, the reagents used in the present invention can be purchased from the market.

[0031] Example 1

[0032] A method for preparing a litsea cubeba nanoemulsion, comprising:

[0033] (1) Soak the seeds of Litsea cubeba in methanol in the dark for 72 hours and then filter the mixture. Take the crude methanol extract by vacuum rotary evaporation of the filtrate;

[0034] (2) liquid-liquid extraction of the crude methanol extract, continued vacuum rotary evaporation, and then air-dried to obtain a litsea cubeba petroleum ether extract;

[0035] (3) Take 1 mL of EL-80 and then 1.5 mL of S-200, mix the two evenly, then weigh 1 g of Litsea cubeba extract and dissolve it in the above mixed solvent, stir at 1000 rpm at room temperature for 15 min to mix evenly, which is the oil phase.

[0036] (4) adding the oil phase dropwise into 97 mL of deionized water while stirring, and continuing stirring at 1000 rpm for 20 min after the addition is completed, thereby obtaining a litsea cubeba nanoemulsion.

[0037] Comparative Example 1

[0038] Preparation of Litsea cubeba extract

[0039] (1) Dissolve 3.3 g of crude methanol extract of Litsea cubeba in 75 mL of petroleum ether, then add deionized water in a volume ratio of 1:1 and mix well. Then, let stand at room temperature for 4-8 hours, and extract with a separatory funnel to obtain a liquid petroleum ether extract;

[0040] (2) The obtained liquid petroleum ether extract was subjected to vacuum rotary evaporation, and finally placed in a 9 cm petri dish in a cool and ventilated place to dry, thereby obtaining the Litsea cubeba extract.

[0041] Test Example 1

[0042] Physicochemical Analysis of Litsea cubeba Nanoemulsion

[0043] (1) Nanoemulsion particle size

[0044] The litsea cubeba nanoemulsion prepared in Example 1 was stored at (0±2)°C for 7 days, and the Zeta potential was measured every day to record its changes. At the same time, an appropriate amount of the nanoemulsion was taken every day to dilute the agent 200 times with deionized water, and the average droplet size was measured using a laser particle size analyzer. Three tests were performed to obtain the average value.

[0045] like Figures 1-2 As shown, the average droplet size of the Litsea cubeba nanoemulsion does not change significantly over time (1-7 days). The particle size change range of the nanoemulsion droplets within 7 days is less than 70nm, and the emulsion system is considered stable. Zeta potential represents the surface charge properties of the nanoemulsion. The larger the absolute value, the greater the mutual charge repulsion, which is sufficient to overcome the intermolecular attraction (i.e., van der Waals force and hydrophobic attraction), and the more stable the emulsion system. After being stored at (0±2)℃ for 7 days, the Zeta potential of the Litsea cubeba nanoemulsion changed slightly, but it was relatively stable overall.

[0046] (2) Nanoemulsion microstructure analysis

[0047] A small amount of YJP-2 fluorescent yellow dye was added during the preparation of the litsea cubeba nanoemulsion. The sample was then evenly dropped on a glass slide, and a laser confocal scanning microscope was used to collect a microscopic image of the droplet distribution of the litsea cubeba nanoemulsion.

[0048] The results are as follows Figure 3As shown, the micrograph results of the prepared nanoemulsions show that the nanoemulsions prepared by EL-80 have smaller droplet sizes (except for a very small number of large particles) and are evenly distributed.

[0049] (3) Centrifugal stability of nanoemulsion

[0050] The nanoemulsion sample stored at (0±2)°C for 7 days was placed in a centrifuge tube and then centrifuged at 8000 rpm for 15 min in a 4°C centrifuge. The proportion of the supernatant in the sample after centrifugation was calculated.

[0051] The results are as follows Figure 4 As shown, after centrifugation, no stratification was observed in the nanoemulsion sample of Litsea cubeba stored at (0±2)°C for 7 days, indicating that the emulsion had good stability.

[0052] Test Example 2

[0053] Comparison of insecticidal effects of Litsea cubeba extract in comparative example 1 and Litsea cubeba nanoemulsion in example 1

[0054] The petroleum ether extract of Litsea cubeba obtained in Comparative Example 1 was weighed and dissolved in acetone and deionized water to prepare a 10 mg / mL mother liquor, and then diluted with 0.2% acetone aqueous solution to a gradient mass concentration (10 mg / mL, 5 mg / mL, 2.5 mg / mL), and fully dissolved and uniformly dissolved with an ultrasonic instrument for later use.

[0055] The litsea cubeba nanoemulsion prepared in Example 1 was diluted with deionized water to 10 mg / mL, 5 mg / mL, and 2.5 mg / mL working solutions for later use.

[0056] Acetone solution was used as blank control.

[0057] (1) Contact toxicity of Litsea cubeba extract and Litsea cubeba nanoemulsion

[0058] The contact activity of the extract of Litsea cubeba and the nanoemulsion of Litsea cubeba was determined by the microdrop method. The volume of 0.5-1 μL was dripped on the pronotum of the 3rd instar larvae of the oriental armyworm using a microdropper or a pipette. Ten healthy 3rd instar larvae of the same size were placed in each dish. Each treatment was repeated 3 times, and the number of dead insects was counted after 24, 48 and 72 hours.

[0059] (2) Gastric toxicity of Litsea cubeba extract and Litsea cubeba nanoemulsion

[0060] The stomach poison activity of the two liquid medicines was determined by leaf immersion method. Fresh corn leaves were cut into rectangular leaf discs (4 cm long and 1 cm wide), and the surface was evenly coated with Litsea cubeba extract or Litsea cubeba nanoemulsion, which was the treatment group. The surface of the leaves was evenly coated with acetone solution as the control group. Then, five treated leaf discs were placed in a culture dish with a diameter of 15 cm. After the liquid was air-dried, 10 healthy, uniformly sized 3rd-instar sticky insect larvae were placed in each dish. Before the experiment, the sticky insect larvae in each dish were starved for 12 hours. Each treatment was repeated 3 times, and the number of dead insects was counted after 24, 48 and 72 hours. The specific results are as follows. Figure 5 As shown:

[0061] according to Figure 5 The results showed that the toxicity of Litsea cubeba nanoemulsion to Oriental armyworm was better than that of Litsea cubeba extract. In addition, at the same concentration, the stomach toxicity of Litsea cubeba nanoemulsion (or Litsea cubeba extract) was better than the contact toxicity. In particular, at a concentration of 10 mg / mL, the stomach toxicity of Litsea cubeba nanoemulsion was as high as 63.33%, which has great potential for application as a botanical insecticide.

[0062] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions only describe the principles of the present invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention. These changes and improvements fall within the scope of the present invention. The scope of protection claimed by the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for preparing a litsea cubeba nanoemulsion, comprising: (1) Soak the seeds of Litsea cubeba in methanol in the dark for 72 hours and then filter the mixture. Vacuum evaporate the filtrate to obtain a crude methanol extract for later use. (2) liquid-liquid extraction of the crude methanol extract, continued vacuum rotary evaporation, and air-dried to obtain a litsea cubeba petroleum ether extract; (3) 1 mL of emulsifier EL-80 and 1.5 mL of S-200 aromatic solvent were taken, and the two were mixed evenly. Then, 1 g of petroleum ether extract of Litsea cubeba was weighed and added thereto, and stirred evenly at room temperature to obtain an oil phase for later use; (4) adding the oil phase drop by drop into the water phase while stirring, and continuing to stir after the addition is completed, thereby obtaining a litsea cubeba nanoemulsion.

2. The preparation method according to claim 1, wherein: The stirring conditions at room temperature in step (3) are: The stirring speed was 1000 rpm and the stirring time was 15 min.

3. The preparation method according to claim 1, wherein: The aqueous phase in step (4) is deionized water.

4. The preparation method according to claim 1, wherein: The amount of deionized water used is 97 mL.

5. The preparation method according to claim 1, wherein: The conditions for continuous stirring in step (4) are: The stirring speed was 1000 rpm and the stirring time was 20 min.

6. A litsea cubeba nanoemulsion prepared according to the preparation method according to any one of claims 1 to 5.

7. Use of the litsea cubeba nanoemulsion according to claim 6 in the preparation of a botanical insecticide for controlling oriental armyworm.

8. The use according to claim 7, wherein: In the botanical insecticide, the concentration of the Litsea cubeba nanoemulsion is 10 mg / mL, 5 mg / mL, and 2.5 mg / mL.

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