Preparation method and application of linalool nano emulsifier

Abstract

The invention relates to the technical field of chemical engineering, in particular to a preparation method and application of a linalool nano emulsifier. Comprising the following steps: obtaining materials, and preparing a linalool essential oil nano-emulsion; the average particle size, the PDI value and the Zeta-potential of the prepared linalool essential oil nano-emulsion are measured; analyzing the storage stability and the freeze-thaw stability of the linalool essential oil nano-emulsion; the biological activity of 3-year old migratory locust nymphs is measured by adopting an aromatic essential oil nano-emulsion; by means of the mode, the stable prevention and control effect can be achieved without depending on improvement of the pesticide application concentration or increase of the pesticide application frequency.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of chemical industry, and particularly relates to a preparation method and application of a linalool nano-emulsifier. BACKGROUND

[0002] As an important agricultural pest, the East Asian migratory locust poses a serious threat to global food production. Locusts are an important part of the agroforestry and pastoral ecosystem, and many harmful locusts can cause varying degrees of harm to agriculture, forestry, and animal husbandry. The East Asian migratory locust, as a worldwide agricultural pest, poses a serious threat to food production.

[0003] Currently, most plant essential oils have relatively small molecular weights, high lipid solubility, which also helps the diffusion of drugs, easy to evaporate and volatilize at room temperature, and have a unique aromatic odor. Plant essential oils and their isolated monomer products belong to a class of chemical information substances that can trigger corresponding chemical changes in insects, animals, or plants. According to existing literature, the biological activity of essential oils on insects is usually studied from aspects such as repellency, fumigation, contact killing, and attraction. Therefore, the prevention and control of the East Asian migratory locust mainly relies on chemical pesticides.

[0004] However, excessive use of chemical pesticides can increase the resistance of the East Asian migratory locust, leading to a continuous decline in the insecticidal effect of existing pesticides, which requires continuous improvement of pesticide concentration or increase in pesticide frequency to maintain the control effect, resulting in poor use effect. SUMMARY

[0005] The present application aims to provide a preparation method and application of a linalool nano-emulsifier, which aims to solve the technical problem of excessive use of chemical pesticides in the prior art, which can increase the resistance of the East Asian migratory locust, leading to a continuous decline in the insecticidal effect of existing pesticides, which requires continuous improvement of pesticide concentration or increase in pesticide frequency to maintain the control effect, resulting in poor use effect.

[0006] To achieve the above-mentioned purpose, a preparation method of a linalool nano-emulsifier is adopted, which comprises the following steps:

[0007] Obtaining materials and preparing a linalool essential oil nano-emulsion;

[0008] Determining the average particle size, PDI value, and Zeta potential of the prepared linalool essential oil nano-emulsion;

[0009] Analyzing the storage stability and freeze-thaw stability of the linalool essential oil nano-emulsion;

[0010] Determining the biological activity of the linalool essential oil nano-emulsion on 3rd instar East Asian migratory locust nymphs.

[0011] In the step of obtaining materials and preparing a linalool essential oil nano-emulsion:

[0012] Using a pipette to suck 20 parts of linalool essential oil into a beaker;

[0013] 2.5 parts of surfactant were added dropwise to the linalool essential oil, and the surfactant was composed of Span 80 and Tween 80;

[0014] The linalool essential oil and the surfactant were fully mixed by magnetic stirring with the help of a magnetic stirrer;

[0015] Deionized water was added to 77.5 parts, and a nano homogenizer was used to repeat homogenization twice to obtain a linalool essential oil nanoemulsion.

[0016] In the step of adding 2.5 parts of surfactant dropwise to the linalool essential oil, and the surfactant was composed of Span 80 and Tween 80:

[0017] The selection of the surfactant compound ratio was selected with a total surfactant content of 2.5 parts, and the Span 80 and Tween 80 ratio was set as 0:2, 1:1, 1:2, 2:1, and 2:0. The selection of the surfactant content was selected with a surfactant Span 80 and Tween 80 compound ratio of 1:2, and the surfactant percentage content was selected as 1%, 1.5%, 2%, 2.5%, and 3%.

[0018] In the step of determining the average particle size, PDI value, and Zeta-potential of the prepared linalool essential oil nanoemulsion:

[0019] The linalool essential oil nanoemulsion was diluted 100 times by volume ratio using ultrapure water;

[0020] Under the condition that the refractive index was set to 1.458, the emulsion particle size, polydispersity index, and Zeta-potential were determined, the equilibrium time was set to 120 s, and the determination was repeated 3 times;

[0021] The linalool essential oil nanoemulsion was placed in a centrifuge, set to 3000 r / min for a total of 5 min, after the end, the lower layer sample was sucked, and the absorbance value of the sample at 600 nm wavelength was measured by an enzyme marker.

[0022] In the step of analyzing the storage stability and freeze-thaw stability of the linalool essential oil nanoemulsion:

[0023] The emulsion of the optimal formula was stored in a 4°C refrigerator, the average particle size and Zeta-potential were determined at 0, 10, 20, and 30 d of storage time, the apparent morphology of the linalool essential oil nanoemulsion was observed, and the determination was repeated 3 times;

[0024] The emulsion with the optimal formula was stored in a -20℃ refrigerator for 24h, then taken out, frozen and thawed at room temperature, and the particle size and Zeta potential were determined after 0, 1, 2 and 3 times of freezing and thawing, and the apparent morphology was observed, and the operation was repeated for 3 times.

[0025] In the step of determining the biological activity of the aromatic essential oil nanoemulsion on the 3rd instar nymphs of the migratory locust, the following steps are performed:

[0026] Six kinds of aromatic essential oil nanoemulsions of linalool, terpinolene, 1,8-cineole, alpha-terpineol, myrcene and alpha-pinene are prepared, and the biological activity of the aromatic essential oil nanoemulsion on the 3rd instar nymphs of the migratory locust is determined;

[0027] The enzyme activity in the migratory locust treated with the linalool essential oil nanoemulsion is determined.

[0028] Data processing and analysis are performed.

[0029] In the step of preparing six kinds of aromatic essential oil nanoemulsions of linalool, terpinolene, 1,8-cineole, alpha-terpineol, myrcene and alpha-pinene, and determining the biological activity of the aromatic essential oil nanoemulsion on the 3rd instar nymphs of the migratory locust, the following steps are performed:

[0030] The contact killing activity is determined by the immersion method.

[0031] The repellent activity is determined by the filter paper method.

[0032] The stomach poison activity is determined by the grass immersion method.

[0033] The antifeedant activity is determined by the leaf disc method.

[0034] In the step of determining the enzyme activity in the migratory locust treated with the linalool essential oil nanoemulsion, the following steps are performed:

[0035] The migratory locusts are treated with 0%, 2.5%, 5.0%, 7.5% and 10.0% linalool essential oil nanoemulsion according to the stomach poison activity determination method, and the treatment time is 5d.

[0036] The surviving migratory locusts in the insect rearing box are taken out and placed in a frozen sample tube, then rapidly frozen in liquid nitrogen and stored in a -80℃ ultra-low temperature refrigerator.

[0037] The protective enzyme, AChE, detoxifying enzyme and digestive enzyme activities of the migratory locust are determined according to the enzyme activity determination kit instructions.

[0038] In the step of performing data processing and analysis, the LC 50 and the 95% confidence interval thereof are calculated by SPSS analysis.

[0039] The activity intensity of the essential oil monomer is evaluated by the median lethal concentration and the median antifeedant concentration.

[0040] The SPSS22.0 software is used for statistical analysis of the experimental data;

[0041] The single factor variance analysis is used for comparing the difference significance between different treatment groups, and the difference is considered to be significant when P<0.05;

[0042] Duncan's new multiple comparison method is used for multiple comparisons.

[0043] The application also provides an application of the preparation method of the linalool nanoemulsifier in biological activity determination of 3-year-old locusts.

[0044] The preparation method and application of the linalool nanoemulsifier provided by the application first obtain materials, prepare linalool essential oil nanoemulsion, then determine the average particle size, PDI value and Zeta-potential of the prepared linalool essential oil nanoemulsion, then analyze the storage stability and freeze-thaw stability of the linalool essential oil nanoemulsion, and finally determine the biological activity of the aromatic essential oil nanoemulsion on 3-year-old locust nymphs, so that the stable prevention and control effect can be achieved without relying on increasing the application concentration or increasing the application frequency. BRIEF DESCRIPTION OF DRAWINGS

[0045] In order to more clearly illustrate the technical solutions of the embodiments of the application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0046] Figure 1 is a step flow chart of the preparation method of the linalool nanoemulsifier of the application.

[0047] Figure 2 is a step flow chart of S100 of the application.

[0048] Figure 3 is a step flow chart of S200 of the application.

[0049] Figure 4 is a step flow chart of S300 of the application.

[0050] Figure 5 is a step flow chart of S400 of the application.

[0051] Figure 6 is a step flow chart of S401 of the application.

[0052] Figure 7 is a step flow chart of S402 of the application.

[0053] Figure 8 is a step flow chart of S403 of the present application.

[0054] Figure 9 is a graph of the effect of surfactant complex ratio on emulsion particle size and PDI value of the present application.

[0055] Figure 10 is a graph of the effect of surfactant complex ratio on centrifugal stability and Zeta-potential of the present application.

[0056] Figure 11 is a graph of the effect of surfactant content on emulsion particle size and PDI value of the present application.

[0057] Figure 12 is a graph of the effect of surfactant content on centrifugal stability and Zeta-potential of the present application.

[0058] Figure 13 is a graph of the effect of different storage times on emulsion particle size and Zeta-potential of the present application.

[0059] Figure 14 is the effect of different storage times on the apparent morphology of the present application.

[0060] Figure 15 is a graph of the effect of freeze-thaw cycle number on emulsion particle size and Zeta-potential of the present application.

[0061] Figure 16 is a graph of the effect of freeze-thaw cycle number on the apparent morphology of the present application.

[0062] Figure 17 is a graph of the touch killing activity of linalool essential oil nanoemulsion on Locusta migratoria manilensis of the present application.

[0063] Figure 18 is a graph of the repellency rate of linalool essential oil nanoemulsion on Locusta migratoria manilensis of the present application.

[0064] Figure 19 is a graph of the stomach poisoning activity of linalool essential oil nanoemulsion on Locusta migratoria manilensis of the present application.

[0065] Figure 20 is a graph of the antifeeding activity of linalool essential oil nanoemulsion on Locusta migratoria manilensis of the present application.

[0066] Figure 21 is a graph of the effect of different concentrations of linalool on the protective enzyme activity in Locusta migratoria manilensis of the present application.

[0067] Figure 22 is a graph of the effect of different concentrations of linalool on the detoxification enzyme activity in Locusta migratoria manilensis of the present application.

[0068] Figure 23 is a graph showing the effect of different concentrations of linalool on the AChE activity in the body of Locusta migratoria.

[0069] Figure 24 is a graph showing the effect of different concentrations of linalool on the digestive enzyme activity in the body of Locusta migratoria. DETAILED DESCRIPTION

[0070] The exemplary embodiments will be described in detail herein with reference to the attached drawings. In the following description, the same numbers are used to indicate the same or similar components. The embodiments described in the following exemplary embodiments are not meant to represent all embodiments consistent with the present application.

[0071] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0072] It is to be understood that the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It is to be understood that the term "and / or" as used herein encompasses all possible combinations of one or more of the associated listed items and can be abbreviated as "or". It is further understood that the terms "comprise" and / or "comprising", when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0073] Referring to Figures 1-8 The present application provides a preparation method of linalool nanoemulsifier, comprising the following steps:

[0074] S100: Obtain materials and prepare linalool essential oil nanoemulsion. In this embodiment, the materials are obtained and the linalool essential oil nanoemulsion is prepared, and the specific process is as follows:

[0075] S101: Use a pipette to suck 20 parts of linalool essential oil into a beaker;

[0076] S102: Drop 2.5 parts of surfactant into the linalool essential oil, and the surfactant is composed of Span 80 and Tween 80;

[0077] S103: Use a magnetic stirrer to fully mix the linalool essential oil and the surfactant;

[0078] S104: Add deionized water to 77.5 parts, and homogenize twice using a nano-homogenizer to obtain linalool essential oil nanoemulsion.

[0079] In the above process, firstly, 20 parts of linalool essential oil were pipetted into a beaker, and 2.5 parts of surfactant (Span 80 and Tween 80) were added dropwise to the linalool essential oil. Then, the linalool essential oil and surfactant were thoroughly mixed by magnetic stirring with a magnetic stirrer. Subsequently, deionized water was added to 77.5 parts, and the mixture was homogenized twice using a nano-homogenizer to obtain a linalool essential oil nanoemulsion.

[0080] Among them, the screening of surfactant compounding ratio: the total surfactant content was selected as 2.5 parts, and the ratio of Span 80 and Tween 80 was set as 0:2, 1:1, 1:2, 2:1, 2:0. Different linalool nanoemulsions were prepared according to method 2.1, and repeated 3 times to investigate the effects of various factors on the average particle size, zeta potential, PDI and centrifugal stability of the prepared linalool essential oil nanoemulsion.

[0081] Screening of surfactant content: The ratio of surfactants Span 80 and Tween 80 was selected as 1:2. Different linalool essential oil nanoemulsions were prepared according to method S100 with surfactant contents of 1%, 1.5%, 2%, 2.5%, and 3%, respectively. The mixture was repeated 3 times to investigate the effects of various factors on the average particle size, zeta potential, PDI, and centrifugal stability of the prepared linalool essential oil nanoemulsions.

[0082] S200: The average particle size, PDI value, and Zeta potential of the prepared linalool essential oil nanoemulsion were determined.

[0083] In this embodiment, the average particle size, PDI value, and Zeta-potential of the prepared linalool essential oil nanoemulsion were measured. The specific process is as follows:

[0084] S201: Dilute the linalool essential oil nanoemulsion 100 times by volume using ultrapure water;

[0085] S202: Under the condition of setting the refractive index to 1.458, the particle size, polydispersity index and Zeta potential of the emulsion were measured. The equilibration time was set to 120s and repeated 3 times.

[0086] S203: Place the linalool essential oil nanoemulsion in a centrifuge and centrifuge at 3000 r / min for 5 min. After centrifugation, aspirate the lower layer sample and measure the absorbance of the sample at 600 nm using an ELISA reader.

[0087] In the above process, the linalool essential oil nanoemulsion was first diluted 100 times by volume with ultrapure water.

[0088] Then, under the condition that the refractive index is set to 1.458, the emulsion particle size, polydispersity index and Zeta-potential are measured, the equilibrium time is set to 120s, and the measurement is repeated 3 times;

[0089] The centrifugal stability measurement method is as follows: the linalool essential oil nanoemulsion is placed in a centrifuge, set to 3000r / min for 5min, after the end, the lower sample is taken, the absorbance value of the sample at 600nm wavelength is measured by the enzyme label instrument, and the centrifugal stability constant K is calculated by formula (1):

[0090] K=(A0–A) / A0(1) × 100%(1) In the formula: K is the centrifugal stability constant / %; A0 is the absorbance value of the sample before centrifugation; A is the absorbance value of the lower sample after centrifugation.

[0091] S300: analyze the storage stability and freeze-thaw stability of linalool essential oil nanoemulsion.

[0092] In this embodiment, the storage stability and freeze-thaw stability of linalool essential oil nanoemulsion are analyzed, and the specific process is as follows:

[0093] S301: store the emulsion of the optimal formula in a 4℃ refrigerator, measure the average particle size and Zeta-potential at 0, 10, 20, 30d of storage time, observe the apparent morphology of linalool essential oil nanoemulsion, and repeat 3 times.

[0094] S302: take the emulsion of the optimal formula, store it in a-20℃ refrigerator for 24h, take it out, freeze-thaw at room temperature, measure the particle size and Zeta-potential after 0, 1, 2 and 3 times of freeze-thaw, and observe the apparent morphology, repeat 3 times.

[0095] In the above process, the emulsion of the optimal formula is stored in a 4℃ refrigerator, the average particle size and Zeta-potential at 0, 10, 20, 30d of storage time are measured, the apparent morphology of linalool essential oil nanoemulsion is observed, and the measurement is repeated 3 times; and the emulsion of the optimal formula is stored in a-20℃ refrigerator for 24h, taken out, freeze-thaw at room temperature, the particle size and Zeta-potential are measured after 0, 1, 2 and 3 times of freeze-thaw, and the apparent morphology is observed, and the measurement is repeated 3 times.

[0096] S400: the biological activity of 3rd instar locust nymphs is measured by using the nanoemulsion of aromatic essential oil. In this embodiment, the biological activity of 3rd instar locust nymphs is measured by using the nanoemulsion of aromatic essential oil, and the specific process is as follows:

[0097] S401: Formulate six kinds of aromatic essential oil nanoemulsions of linalool, terpinolene, 1,8-cineole, alpha-terpineol, myrcene and alpha-pinene, and determine the biological activity of the aromatic essential oil nanoemulsion on 3rd instar locust nymphs;

[0098] S402: Determine the enzyme activity in the body of the locusts for the linalool essential oil nanoemulsion;

[0099] S403: Perform data processing and analysis.

[0100] In the above process, first, six kinds of aromatic essential oil nanoemulsions of linalool, terpinolene, 1,8-cineole, alpha-terpineol, myrcene and alpha-pinene are prepared, then the enzyme activity in the body of the locusts for the linalool essential oil nanoemulsion is determined, and then data processing and analysis are performed, by preparing six kinds of aromatic essential oil nanoemulsions of linalool, terpinolene, 1,8-cineole, alpha-terpineol, myrcene and alpha-pinene.

[0101] In this embodiment, six kinds of aromatic essential oil nanoemulsions of linalool, terpinolene, 1,8-cineole, alpha-terpineol, myrcene and alpha-pinene are prepared, and the biological activity of the aromatic essential oil nanoemulsion on 3rd instar locust nymphs is determined, and the specific process is as follows:

[0102] S4011: Determine the contact toxicity by the dipping method;

[0103] S4012: Determine the repellent activity by the filter paper method;

[0104] S4013: Determine the stomach toxicity by the grass dipping method;

[0105] S4014: Determine the antifeedant activity by the leaf disc method.

[0106] In the above process, the contact toxicity is determined by the dipping method, according to the pre-test results, the locusts are immersed in five different concentrations of essential oil nanoemulsions for 10s, then placed in a feeding cage containing fresh wheat leaves, and fed at 26±2℃, and the mortality of the locusts is calculated according to formulas (2) and (3) at 6h, 12h and 24h, and the lethal concentration of the essential oil nanoemulsion is obtained by SPSS analysis, 10 locusts are treated at each concentration, and the process is repeated 6 times,

[0107] Mortality (%) = number of dead insects / total number of treated insects × 100% (2)

[0108] Corrected mortality (%) = [(treatment group mortality-control group mortality) / (1-control group mortality)] × 100% (3);

[0109] The repellent activity was determined by using filter paper method. 500 μL of essential oil nanoemulsion of five different concentrations was dropped on one side of a 9 cm filter paper, and the same amount of control solution was dropped on the other side. Locusta migratoria migratoria was placed in the middle of the 9 cm filter paper, and the selection of the locusts within 30 min was recorded. The repellency rate was calculated according to formula (4), 10 locusts were treated at each concentration, and the test was repeated three times,

[0110] Repellency rate (%) = (control insect number - treatment insect number) / (control insect number + treatment insect number) x 100% (4);

[0111] The stomach poison activity was determined by using the grass immersion method. Fresh wheat leaves were immersed in essential oil nanoemulsion of five different concentrations for 10 s, taken out and ventilated, and then used to feed Locusta migratoria migratoria. The survival of Locusta migratoria migratoria was observed and recorded at 24 h, 48 h and 72 h. The mortality and corrected mortality of test insects were calculated according to calculation formulas (2) and (3), and LC 50 ;

[0112] The antifeedant activity was determined by using the leaf disc method. According to the pre-experiment, five different concentrations of essential oil nanoemulsion were evenly sprayed on the leaf disc surface of wheat leaves. After the solvent was volatilized, the leaf disc was placed in a culture dish. One Locusta migratoria migratoria starved for 24 h was placed in the culture dish. Ten locusts were treated at different concentrations. After 36 h, the remaining area of the leaf disc eaten by Locusta migratoria migratoria was measured. The antifeedant concentration (AFC 50 ) of each essential oil was calculated according to formula (5). Each treatment was set with three replicates. The non-selective antifeedant rate (%) = [(control group leaf area eaten - treatment group leaf area eaten) / control group leaf area eaten] x 100% (5).

[0113] S402: Determination of linalool essential oil nanoemulsion on the enzyme activity in Locusta migratoria migratoria. In this embodiment, the linalool essential oil nanoemulsion was determined on the enzyme activity in Locusta migratoria migratoria. The specific process is as follows:

[0114] S4021: Select 0%, 2.5%, 5.0%, 7.5%, 10.0% linalool essential oil nanoemulsion to treat Locusta migratoria migratoria according to the determination method of stomach poison activity. The treatment time is 5 d;

[0115] S4022: Take out the surviving Locusta migratoria migratoria in the insect rearing box and place it in a frozen sample tube. After rapid freezing in liquid nitrogen, store it in a -80℃ ultra-low temperature refrigerator;

[0116] S4023: Refer to the instruction manual of the enzyme activity determination kit to determine the protective enzyme, AChE, detoxifying enzyme and digestive enzyme activity of Locusta migratoria migratoria.

[0117] In the above process, 0%, 2.5%, 5.0%, 7.5%, 10.0% linalool essential oil nanoemulsion is selected according to the determination of stomach poison activity to treat locust, and the locusts surviving in the incubation box after 5 days are taken out and placed in a frozen sample tube, then rapidly frozen in liquid nitrogen and stored in a-80℃ ultra-low temperature refrigerator, and the protective enzyme (SOD, CAT, POD) activity, AChE, detoxification enzyme (GST, MFO, CYP450 and CES), digestive enzyme (AMS, lipase, trypsin) activity of the locust are determined according to the enzyme activity determination kit instruction.

[0118] S403: data processing and analysis are performed. In this embodiment, data processing and analysis are performed, and the specific process is:

[0119] S4031: LC 50 and the 95% confidence interval thereof are calculated by SPSS analysis.

[0120] S4032: the activity intensity of the essential oil monomer is evaluated by the median lethal concentration and the median antifeeding concentration.

[0121] S4033: the test data are statistically analyzed by using SPSS22.0 software.

[0122] S4034: the difference significance between different treatment groups is compared by using one-way analysis of variance, and the difference is considered to be significant when P<0.05.

[0123] S4035: Duncan's new multiple range method is used for multiple comparison.

[0124] In the above process, for the determination of contact killing, repellence, stomach poisoning and antifeeding activity, LC 50 and the 95% confidence interval thereof are calculated by SPSS analysis, and the activity intensity of the essential oil monomer is evaluated by the median lethal concentration (LC 50 ), the median antifeeding concentration (AFC 50 ). All test data are statistically analyzed by using SPSS22.0 software, and the difference significance between different treatment groups is compared by using one-way analysis of variance (One-way ANOVA), and the difference is considered to be significant when P<0.05, and then Duncan's new multiple range method is used for multiple comparison. The difference between the test group and the control group is evaluated by using T test for enzyme activity analysis.

[0125] Further, the application provides a preparation method of the linalool nanoemulsifier, which further comprises result analysis.

[0126] Single factor investigation of the linalool essential oil nanoemulsion formula: the screening of the compound ratio of the surfactant is carried out by Figure 9It can be seen that the particle size of the test group with the ratio of Span 80 to Tween 80 of 1:2 and 2:1 was significantly higher than that of the test group with the ratio of 1:1 and 0:2 (P<0.05), indicating that the increase of surfactant in this system is conducive to the formation of nanoemulsion. The PDI of the test group with the ratio of Span 80 to Tween 80 of 1:1 and 1:2 was significantly lower than that of other test groups (P<0.05). Studies have shown that the smaller the PDI value, the more uniform the particle size distribution of the emulsion. From the above, Figure 10 It can be seen that the Zeta-potential of each complex ratio emulsion was stably maintained at about -6 mV, and there was no significant difference (P>0.05), which may be due to the fact that Span 80 and Tween 80 are both non-ionic surfactants and do not ionize into ions (uncharged) in aqueous solution. Therefore, the change of the ratio of the two reagents does not affect the Zeta-potential of the emulsion. When the ratio of surfactant Span 80 to Tween 80 is 0:2 and 2:0, the centrifugal stability of the emulsion is the lowest. In summary, through the indicators of particle size, PDI, Zeta-potential and centrifugal stability, the ratio of Span 80 to Tween 80 of 1:2 is more appropriate;

[0127] The selection of surfactant content is from Figure 11 It can be seen that when the surfactant content (the ratio of Span 80 to Tween 80 is 1:2) is 1.5%, the particle size of the emulsion is significantly lower than that of other content groups (P<0.05), and the particle size of the 2.5% group has no significant difference with that of the 2.0% and 3.0% groups (P>0.05). From the PDI, the PDI of the 2.5% content group is significantly lower than that of the 1.0%, 1.5% and 2.0% groups (P<0.05), and has no significant difference with that of the 3.0% content group (P>0.05). From the Zeta-potential, Figure 12 It can be seen that, similar to the selection of the complex ratio, the Zeta-potential of each surfactant content emulsion is stably maintained at about -6 mV, and there is no significant difference (P>0.05). When the surfactant content is 2.5%, the centrifugal stability is significantly higher than that of other content groups. In summary, through the indicators of particle size, PDI, Zeta-potential and centrifugal stability, the surfactant content of 2.5% is more appropriate.

[0128] Stability analysis of linalool essential oil nanoemulsion:

[0129] The storage stability of linalool essential oil nanoemulsion is analyzed by Figure 13 It can be seen that with the extension of storage time, there is no significant difference between the particle size and Zeta-potential of the emulsion of 0, 10, 20 and 30 days (P>0.05), and the Zeta-potential is stably maintained at -6.0 mV to -7.0 mV. From the PDI, Figure 14The results showed that a storage time of 30 days did not affect the stability of the emulsion, and no stratification or demulsification occurred. Therefore, the linalool essential oil nanoemulsion exhibits good storage stability.

[0130] The freeze-thaw stability of linalool essential oil nanoemulsion was assessed through... Figure 15 It can be seen that with the increase of freeze-thaw cycles 0, 1, 2, and 3, there were no significant differences in the particle size and zeta potential of the emulsion (P > 0.05). The particle size remained between 120 and 140 nm, and the zeta potential remained stable between -6.4 mV and -7.4 mV. Figure 16 The results show that three freeze-thaw cycles do not affect the stability of the emulsion, and no stratification or demulsification occurred. Therefore, the linalool essential oil nanoemulsion exhibits good freeze-thaw stability.

[0131] Bioactivity of essential oil nanoemulsions on 3rd instar nymphs of the Oriental migratory locust:

[0132] Contact activity assay, by Figure 17 It was found that all essential oils possessed certain contact toxicity against the Oriental migratory locust, and the lethal median dose (LC50) decreased with prolonged treatment time. 1,8-Cineole, linalool, and myrcene exhibited the strongest contact toxicity, with corresponding LC50 values ​​at a treatment time of 6 hours. 50 The concentrations were 2.93%, 3.58%, and 4.01%, respectively, with no significant difference between 1,8-cineole and linalool. At treatment times of 12 and 24 hours, myrcene was more effective than the other two.

[0133] Repellent activity assay, by Figure 18 The results showed that the repellency of the six essential oils against the migratory locusts increased first and then stabilized with increasing concentration. Among them, linalool and α-terpineol showed the strongest repellency against the migratory locusts, with no significant difference between the two. 1,8-cineole had the worst repellency effect.

[0134] Gastric toxicity assay, by Figure 19 As shown, at an action time of 24 hours, the concentrations of isoprene, 1,8-cineole, myrcene, and α-pinene essential oils did not exhibit stomach poison activity against the Oriental migratory locust, while linalool showed stronger stomach poison activity. (LC50 data is missing from the original text.) 50 The LC50 was 27.36%, followed by α-terpineol. 50 The effect rate was 41.82%. At an action time of 48 hours, myrcene showed strong gastric toxicity activity, which was stronger than that of linalool. However, at an action time of 72 hours, the gastric toxicity activity from strongest to weakest was linalool, myrcene, α-terpineol, α-pinene, isoterpinene, and 1,8-cineole.

[0135] The determination of anorexia activity was performed by measuring the table. Figure 20 As shown, 1,8-cineole and linalool exhibited strong antifeedant activity, with corresponding AFC values ​​of [missing information].50 The antifeedant effect of alpha-pinene and myrcene was not obvious, and the antifeedant medium concentration was 3.27% and 3.44% respectively.

[0136] Determination of enzyme activity in the body of locusts treated with linalool nanoemulsion:

[0137] Determination of protective enzyme activity, locusts were treated with different concentrations of linalool, and the CAT, SOD and POD activities in the body of the locusts were determined. Figure 21 It can be known that with the increase of the concentration of the essential oil, the CAT and POD in the body of the locusts were first activated and then inhibited, and the POD was activated again at the concentration of 10% after being inhibited.

[0138] Determination of AChE and detoxification enzyme activity, locusts were treated with different concentrations of linalool, and the AChE, detoxification enzyme (CYP450, GST, CES, MFO) activities in the body of the locusts were determined. Figure 22 It can be known that with the increase of the concentration of the essential oil, the CAT and POD in the body of the locusts were first activated and then inhibited, and the POD was activated again at the concentration of 10% after being inhibited. Figure 23 It can be known that with the increase of the concentration of the essential oil, the CAT and POD in the body of the locusts were first activated and then inhibited, and the POD was activated again at the concentration of 10% after being inhibited.

[0139] Determination of digestive enzyme activity, locusts were treated with different concentrations of linalool, and the AMS, lipase and trypsin activities in the body of the locusts were determined. Figure 24 It can be known that the treatment of the essential oil inhibited the lipase activity in the body of the locusts and activated the AMS activity.

[0140] The application further provides application of the preparation method of the linalool nanoemulsifier in determination of the biological activity of the 3rd instar locust.

[0141] Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. It is intended that the application be limited only by the scope of the claims, including any amendments thereof, and other equivalents to the claims. It is intended that the application not be limited to the examples and embodiments described herein, but that the application include any and all embodiments within the scope of the claims.

[0142] It should be understood that the application is not limited to the precise construction that has been described above and illustrated in the accompanying drawings and that various modifications and changes can be made by those skilled in the art without departing from the scope of the application.

Claims

1. A method for preparing a linalool nanoemulsifier, characterized by, The method comprises the following steps: Obtaining materials, preparing linalool essential oil nanoemulsion; Measuring the average particle size, PDI value and Zeta potential of the prepared linalool essential oil nanoemulsion; Analyzing the storage stability and freeze-thaw stability of the linalool essential oil nanoemulsion; Determining the biological activity of the linalool essential oil nanoemulsion on 3rd instar locust nymphs.

2. The method for preparing linalool nanoemulsifier as described in claim 1, characterized in that, In the step of obtaining materials and preparing linalool essential oil nanoemulsion: Use a pipette to suck 20 parts of linalool essential oil into a beaker; Drop 2.5 parts of surfactant into the linalool essential oil, and the surfactant is composed of Span 80 and Tween 80; Use a magnetic stirrer to fully mix the linalool essential oil and the surfactant; Add deionized water to 77.5 parts, and use a nano homogenizer to repeat homogenization twice to obtain linalool essential oil nanoemulsion.

3. The method for preparing linalool nanoemulsifier as described in claim 2, characterized in that, In the step of dropping 2.5 parts of surfactant into the linalool essential oil, and the surfactant is composed of Span 80 and Tween 80: Screening of surfactant compound ratio, selecting the total content of surfactant as 2.5 parts, setting the ratio of Span 80 and Tween 80 as 0:2, 1:1, 1:2, 2:1 and 2:0; screening of surfactant content, selecting the compound ratio of surfactant Span 80 and Tween 80 as 1:2, and selecting the percentage content of surfactant as 1%, 1.5%, 2%, 2.5% and 3%.

4. The method of claim 1, wherein the preparation of the linalool nanoemulsifier is characterized by, In the step of measuring the average particle size, PDI value and Zeta potential of the prepared linalool essential oil nanoemulsion: Use ultrapure water to dilute the linalool essential oil nanoemulsion by 100 times in volume ratio; Under the condition that the refractive index is set to 1.458, measure the emulsion particle size, polydispersity index and Zeta potential, and set the equilibrium time to 120s, and repeat 3 times; Place the linalool essential oil nanoemulsion in a centrifuge, set the centrifugation speed to 3000r / min for 5min, after the end, suck the lower sample, and use an enzyme marker to measure the absorbance value of the sample at 600nm wavelength.

5. The method for preparing linalool nanoemulsifier as described in claim 1, characterized in that, In the step of analyzing the storage stability and freeze-thaw stability of the linalool essential oil nanoemulsion: Store the emulsion of the optimal formula in a 4℃ refrigerator, measure the average particle size and Zeta potential of the emulsion stored for 0, 10, 20 and 30d, observe the apparent morphology of the linalool essential oil nanoemulsion, and repeat 3 times; Store the emulsion of the optimal formula in a-20℃ refrigerator for 24h, take it out, freeze-thaw it under room temperature conditions, measure the particle size and Zeta potential after freeze-thawing for 0, 1, 2 and 3 times, and observe the apparent morphology, and repeat 3 times.

6. The method for preparing linalool nanoemulsifier as described in claim 1, characterized in that, In the step of determining the biological activity of the linalool essential oil nanoemulsion on 3rd instar locust nymphs: Prepare 6 kinds of aromatic essential oil nanoemulsions of linalool, isopinocamphone, 1,8-eucalyptol, α-terpineol, myrcene and α-pinene, and determine the biological activity of the aromatic essential oil nanoemulsion on 3rd instar locust nymphs; Determine the enzyme activity of the linalool essential oil nanoemulsion in the locust; Perform data processing and analysis.

7. The method for preparing linalool nanoemulsifier as described in claim 6, characterized in that, In the preparation of linalool, isoeugenol, 1,8-cineole, alpha-terpineol, myrcene and alpha-pinene six kinds of aromatic essential oil nanoemulsion, the steps of measuring the biological activity of the three instar locust nymphs of the aromatic essential oil nanoemulsion are: The dipping method is used to measure the contact activity; The test paper method is used to measure the repellent activity; The grass dipping method is used to measure the stomach poison activity; The leaf disc method is used to measure the antifeedant activity.

8. The method of claim 6, wherein the preparation of the linalool nanoemulsifier is characterized by, In the step of measuring the enzyme activity of linalool essential oil nanoemulsion in the body of locust: Select 0%, 2.5%, 5.0%, 7.5%, 10.0% linalool essential oil nanoemulsion to treat locust according to the method of stomach poison activity determination, and the treatment time is 5d; Take out the surviving locusts in the insect rearing box and place them in a frozen sample tube, then quickly freeze them in liquid nitrogen and store them in a-80℃ ultra-low temperature refrigerator; According to the reagent kit instruction of enzyme activity determination, the protective enzyme, AChE, detoxifying enzyme and digestive enzyme activity of locust are determined.

9. The method for preparing linalool nanoemulsifier as described in claim 6, characterized in that, In the step of data processing and analysis LC was calculated by SPSS analysis 50 and its 95% confidence interval; The activity intensity of essential oil monomer is evaluated by the median lethal concentration and the median antifeedant concentration; The SPSS22.0 software is used to statistically analyze the experimental data; Single factor variance analysis is used to compare the significant difference between different treatment groups, and when P<0.05, it is considered that the difference is significant; Duncan's new multiple range method is used for multiple comparisons.

10. The application of the preparation method of linalool nanoemulsifier in the biological activity determination of the three instar locusts.

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