阅读说明：本技术 一种具有豇豆蚜杀虫活性的柚子油及其杀虫剂和应用 (Shaddock oil with cowpea aphid insecticidal activity, insecticide and application thereof ) 是由 张军 凌微 张婧怡 钟八莲 于 2021-06-24 设计创作，主要内容包括：本发明提供了一种具有豇豆蚜杀虫活性的柚子油及其杀虫剂和应用,属于生物杀虫剂技术领域。本发明提供的柚子油,包含21种主要活性物质,其中D﹣柠檬烯、(Z,Z)﹣2,4﹣癸二烯醛、棕榈酸、亚油酸含量较高(相对含量达10％以上)。本发明通过豇豆蚜触杀和胃毒实验结果表明,所述柚子油对豇豆蚜具有很强的杀虫活性,在触杀浓度为2.0μg/每只虫或胃毒浓度为20mg/mL时,作用72h后,豇豆蚜死亡率分别达到96.5％和93.3％。同时柚子油是从柚子副产物柚子籽中提取得到的植物次生代谢物,属于天然活性物质,具有绿色环保、环境友好、低毒等特点,对人类健康不会造成影响,为新型环保杀虫剂。(The invention provides shaddock oil with cowpea aphid insecticidal activity, and an insecticide and application thereof, and belongs to the technical field of biological insecticides. The pomelo oil provided by the invention contains 21 main active substances, wherein the contents of D-limonene, (Z, Z) -2, 4-decadienal, palmitic acid and linoleic acid are higher (the relative content is more than 10%). According to the experimental results of the contact killing and stomach toxicity of the cowpea aphids, the shaddock oil has strong insecticidal activity on the cowpea aphids, and when the contact killing concentration is 2.0 mu g/each insect or the stomach toxicity concentration is 20mg/mL, the death rates of the cowpea aphids reach 96.5% and 93.3% after 72 hours of action. Meanwhile, the pomelo oil is a plant secondary metabolite extracted from the pomelo by-product pomelo seeds, belongs to a natural active substance, has the characteristics of environmental protection, environmental friendliness, low toxicity and the like, does not influence human health, and is a novel environment-friendly pesticide.)

1. The pomelo oil with the cowpea aphid insecticidal activity is characterized by comprising the following active ingredients: d-limonene, (E) -2-decenal, (E, Z) -2, 4-decadienal, (Z, Z) -2, 4-decadienal, tetradecene, beta-caryophyllene, yanogene, hexadecene, (Z, E) -6, 9-heptadecadiene, 8-heptadecene, octadecene, nootkatone, phytone, methyl palmitate, palmitic acid, ethyl palmitate, methyl linoleate, methyl 10-octadecenoate, linoleic acid, ethyl linoleate and ethyl oleate.

2. The pomelo oil with cowpea aphid insecticidal activity according to claim 1, which is characterized by comprising the following active ingredients in mass content: 22.86% of D-limonene, 1.18% (E) 2-decenal, 7.77% (E, Z) 2, 4-decadienal, 12.40% (Z, Z) 2, 4-decadienal, 0.64% of tetradecene, 1.47% of beta-caryophyllene, 3.38% of elemene, 0.65% of hexadecene, 1.28% (Z, Z) 6, 9-heptadecadiene, 0.83% of 8-heptadecene, 0.71% of octadecene, 1.18% of nootkatone, 0.71% of phytone, 1.11% of methyl palmitate, 15.79% of palmitic acid, 2.42% of ethyl palmitate, 1.05% of methyl linoleate, 0.52% of 10-octadecenoic acid methyl ester, 20.21% of linoleic acid, 1.88% of ethyl linoleate and 1.97% of oleic acid ethyl ester.

3. A process for the preparation of pomelo oil with insecticidal activity against cowpea aphid according to claim 1 or 2, characterized by comprising the steps of:

1) cutting grapefruit seeds into pieces, soaking the grapefruit seeds in an ethanol water solution with the volume concentration of 85-100%, and heating and refluxing the obtained soaking solution for extraction to obtain an extracting solution;

2) removing the solvent of the extracting solution to obtain an extract;

3) suspending the extract with water, extracting the obtained extract suspension aqueous solution with petroleum ether, collecting the petroleum ether layer, and removing the solvent to obtain the pomelo oil.

4. The preparation method according to claim 3, wherein the pomelo seeds soaked in the step 1) are soaked again in the ethanol water solution with the volume concentration of 85-100%, the obtained soaking solution is heated and refluxed for extraction, and the obtained extracting solutions are combined.

5. The preparation method according to claim 3 or 4, wherein the soaking time in the step 1) comprises 10-14 h;

in the step 2), the volume ratio of the extract water solution to the petroleum ether is 1 (0.9-1.1).

6. A pesticide suitable for cowpea aphids, which is characterized in that the pomelo oil of claim 1 or 2 or the pomelo oil prepared by the preparation method of any one of claims 3 to 5 is used as an active ingredient.

7. The cowpea aphid-suitable pesticide according to claim 6, wherein the formulation of the pesticide comprises an emulsifier;

the emulsifier is Tween 80-pomelo oil emulsifier with volume concentration of 0.1%.

8. Use of a grapefruit oil according to claim 1 or 2 or a cowpea aphid-suitable insecticide according to claim 6 or 7 for controlling cowpea aphids.

9. Use of a grapefruit oil according to claim 1 or 2 or a cowpea aphid-suitable insecticide according to claim 6 or 7 for reducing aphid protease activity.

10. The use according to claim 9, wherein the aphid proteases comprise acetylcholinesterase, glutathione S-transferase and peroxidase.

Technical Field

The invention belongs to the technical field of biological insecticides, and particularly relates to pomelo oil with cowpea aphid insecticidal activity, an insecticide of the pomelo oil and application of the pomelo oil.

Background

Pests cause extensive damage to grain crops such as grains and legumes by directly eating plant parts such as shoots, seeds, flowers and plant juices. Pests feed on plants, and their waste accumulation is harmful to plants and human beings that it eats, and at the same time, pests cause crop quality degradation, causing enormous economic losses to farmers. Nearly 5% to 30% of the total annual global agricultural production is reported to be infected with pests.

Cowpea aphids or black bean aphids are common in temperate and tropical regions all over the world, are polyphagic pests with great harm, feed on more than 80 families of plants, and preferentially select leguminosae. Meanwhile, cowpea aphids also seriously damage other important economic crops, such as peanuts, kidney beans, large-leaf kidney beans, common beans and the like. Aphids, one of the most destructive pests on earth, are widely distributed and suck plant juices with sharp mouthparts, causing deterioration of plant nutrition, even defoliation and wilting. Moreover, it can transmit various plant viruses and cause serious economic loss, and the aphid has super-strong reproductive capacity and extremely high transmission speed, and can cause the destruction of the whole vegetable field if not interfered. The use of chemical synthetic insecticides is the main measure for preventing and controlling cowpea aphids at present, however, the use of a large amount of chemical insecticides can enhance the drug resistance of the aphids, bring a large amount of pesticide residues, pollute soil and nearby water areas and seriously affect human health.

In view of the high toxicity and difficult degradability of the chemical synthetic pesticide, in recent years, many researchers at home and abroad are looking for natural and environment-friendly botanical pesticides. Liuzhiping et al studied the insecticidal effect of wintergreen oil on Nilaparvata lugens, and conducted an in-depth study on the insecticidal mechanism of the oil, and the results showed that wintergreen oil can significantly affect the acetylcholinesterase activity in the test insects. Studies of Chenyijuan and the like find that the salvia miltiorrhiza volatile oil has obvious insecticidal activity on beet armyworm, the content of alpha-haematoxylin is the highest through GC-MS detection and analysis, and the compound possibly having the main insecticidal activity is the alpha-haematoxylin. Due to the advantages of high efficiency, low toxicity and definite action target, botanical insecticides have become a research hotspot at present. The literature research shows that at present, no relevant reports about the cowpea aphid killing activity of the grapefruit seed oil and the active chemical components of the grapefruit seed oil exist, and the plant-derived pesticide with the significant insecticidal effect on the cowpea aphids is obtained, so that a green and environment-friendly novel pesticide is provided for agricultural prevention and control of the cowpea aphids. Meanwhile, the high-valued development and utilization of the grapefruit seed byproduct resources are realized powerfully, and the development and the promotion of the grapefruit industrial chain are promoted.

Disclosure of Invention

In view of the above, the invention aims to provide the pomelo oil with the cowpea aphid insecticidal activity, the insecticide and the application thereof, and the pomelo oil has the advantages of environmental protection, environmental friendliness, low toxicity and no side effect on human health.

The invention provides pomelo oil with cowpea aphid insecticidal activity, which comprises the following active ingredients: d-limonene, (E) -2-decenal, (E, Z) -2, 4-decadienal, (Z, Z) -2, 4-decadienal, tetradecene, beta-caryophyllene, yanogene, hexadecene, (Z, E) -6, 9-heptadecadiene, 8-heptadecene, octadecene, nootkatone, phytone, methyl palmitate, palmitic acid, ethyl palmitate, methyl linoleate, methyl 10-octadecenoate, linoleic acid, ethyl linoleate and ethyl oleate.

Preferably, the active ingredients comprise the following mass contents: 22.86% of D-limonene, 1.18% (E) 2-decenal, 7.77% (E, Z) 2, 4-decadienal, 12.40% (Z, Z) 2, 4-decadienal, 0.64% of tetradecene, 1.47% of beta-caryophyllene, 3.38% of elemene, 0.65% of hexadecene, 1.28% (Z, Z) 6, 9-heptadecadiene, 0.83% of 8-heptadecene, 0.71% of octadecene, 1.18% of nootkatone, 0.71% of phytone, 1.11% of methyl palmitate, 15.79% of palmitic acid, 2.42% of ethyl palmitate, 1.05% of methyl linoleate, 0.52% of 10-octadecenoic acid methyl ester, 20.21% of linoleic acid, 1.88% of ethyl linoleate and 1.97% of oleic acid ethyl ester.

The invention provides a preparation method of shaddock oil with cowpea aphid insecticidal activity, which comprises the following steps:

1) cutting grapefruit seeds into pieces, soaking the grapefruit seeds in an ethanol water solution with the volume concentration of 85-100%, and heating and refluxing the obtained soaking solution to obtain an extracting solution;

2) removing the solvent of the extracting solution to obtain an extract;

3) suspending the extract with water, extracting the obtained extract suspension aqueous solution with petroleum ether, collecting petroleum ether layer, and concentrating to obtain pomelo oil.

Preferably, the pomelo seeds soaked in the step 1) are soaked again in an ethanol water solution with the volume concentration of 85-100%, the obtained soaking solution is heated and refluxed for extraction, and the obtained extracting solutions are combined.

Preferably, the soaking time in the step 1) comprises 10-14 hours;

in the step 2), the volume ratio of the extract water solution to the petroleum ether is 1 (0.9-1.1).

The invention provides a pesticide suitable for cowpea aphids, which takes the pomelo oil or the pomelo oil prepared by the preparation method as an active ingredient.

Preferably, the formulation of the pesticide includes an emulsifier;

the emulsifier is Tween 80-pomelo oil emulsifier with volume concentration of 0.1%.

The invention provides application of the shaddock oil or the pesticide applicable to cowpea aphids in prevention and control of the cowpea aphids.

The invention provides application of the shaddock oil or the pesticide applicable to the cowpea aphids in reducing the activity of aphid protease.

Preferably, the aphid proteases comprise acetylcholinesterase, glutathione S-transferase and peroxidase.

The invention provides shaddock oil with cowpea aphid insecticidal activity, which comprises 21 main active substances, wherein the contents of D-limonene, (Z, Z) -2, 4-decadienal, palmitic acid and linoleic acid are higher (the relative content is more than 10%). According to the invention, the results of the contact killing and stomach toxicity tests of the cowpea aphids show that the shaddock oil has stronger insecticidal activity on the cowpea aphids, and the death rate of the cowpea aphids after 72 hours of action reaches 96.5 percent or 93.3 percent when the contact killing concentration is 2.0 mu g/insect or the stomach toxicity concentration is 20 mg/mL. Meanwhile, the plant secondary metabolite extracted from the pomelo seeds by the pomelo oil belongs to natural active substances, has the characteristics of environmental protection, environmental protection and low toxicity, does not influence human health, and provides a new direction for preparing the plant-friendly pesticide.

The invention also provides application of the shaddock oil or the pesticide applicable to the cowpea aphids in prevention and control of the cowpea aphids. The pomelo oil or the pesticide can reduce the activities of acetylcholinesterase, glutathione S-transferase and peroxidase and reduce the content of total protein in the body of the insect, thereby achieving the purpose of killing insects. Experiments show that the pesticide has strong insecticidal effect no matter the pesticide is applied by contact killing or stomach toxicity, the insecticidal effect is positively correlated with the dosage of the pesticide, and the semilethal concentration LC of contact killing activity with 72h of action₅₀1.75 mu g/insect, and the stomach toxicity activity half lethal concentration LC tested by a drug membrane method after 72 hours of drug administration₅₀It was 3.96 mg/mL.

Drawings

FIG. 1 shows GC-MS measurement spectrum results in examples of the present invention;

FIG. 2 shows the results of the determination of the contact killing of the grapefruit oil pesticide on cowpea aphids in the embodiment of the invention;

FIG. 3 shows the result of measuring the stomach toxicity of the grapefruit oil pesticide on cowpea aphid in the embodiment of the invention;

FIG. 4 shows the results of determination of total protein in cowpea aphid by the grapefruit oil pesticide in the embodiment of the invention;

FIG. 5 shows the results of measuring the activity of a grapefruit oil pesticide on acetylcholinesterase in cowpea aphids in the embodiment of the invention;

FIG. 6 shows the measurement result of the glutathione-S-transferase activity of the grapefruit oil pesticide in cowpea aphid according to the embodiment of the invention;

FIG. 7 shows the result of determining peroxidase activity of shaddock oil pesticide in cowpea aphid according to the embodiment of the present invention.

Detailed Description

The invention provides a preparation method of shaddock oil with cowpea aphid insecticidal activity, which comprises the following steps:

1) cutting grapefruit seeds into pieces, soaking the grapefruit seeds in an ethanol water solution with the volume concentration of 85-100%, and heating and refluxing the obtained soaking solution for extraction to obtain an extracting solution;

2) removing the solvent of the extracting solution to obtain an extract;

3) suspending the extract with water, extracting the obtained extract suspension aqueous solution with petroleum ether, collecting the petroleum ether layer, and removing the solvent to obtain the pomelo oil.

The method comprises the steps of cutting grapefruit seeds into pieces, soaking the grapefruit seeds in an ethanol water solution with the volume concentration of 85-100%, and heating, refluxing and extracting the obtained soak solution to obtain an extracting solution.

The source of the grapefruit seeds is not particularly limited in the present invention, and grapefruit seeds of a grapefruit type well known in the art may be used. In the embodiment of the invention, the shaddock seeds are Shatian shaddock seeds. The grapefruit seeds are preferably air-dried grapefruit seeds. The water content of the air-dried grapefruit seeds is not higher than 5%. The degree of cutting of the grapefruit seeds is not particularly limited in the present invention, and the degree of cutting known in the art can be adopted, for example, the particle size of the cut grapefruit seeds is not more than 3 mm.

In the present invention, the volume concentration of the ethanol aqueous solution is preferably 90% to 95%, more preferably 95%. The material-liquid ratio of the grapefruit seeds to the ethanol aqueous solution is 1 g: (1-1.25) ml. The soaking time is preferably 10-14 h, and more preferably 12 h. The temperature of the soaking is not particularly limited in the present invention, and the soaking temperature known in the art, for example, room temperature, may be used. The heating reflux temperature is preferably 78-82 ℃, and more preferably 80 ℃. The heating reflux time is preferably 1.5-3 h, and more preferably 2-2.5 h.

In the invention, the soaked pomelo seeds are preferably soaked again by using ethanol water solution with the volume concentration of 85-100%, the obtained soaking solution is heated and refluxed for extraction, and the obtained extracting solutions are combined. The soaking and heating reflux schemes are the same as those described above, and are not described herein.

After the extracting solution is obtained, the solvent of the extracting solution is removed to obtain the extract.

In the present invention, the solvent method for removing the leachate preferably comprises concentration under reduced pressure. The present invention is not particularly limited in the scheme of the concentration, and a concentration scheme well known in the art, such as concentration under reduced pressure, may be employed. The pressure of the reduced pressure concentration is 0.1Mpa, and the temperature of the reduced pressure concentration is preferably 42-47 ℃, and more preferably 45 ℃. The time for the reduced pressure concentration is 3-5 h, and more preferably 4 h.

After obtaining the extract, suspending the extract in water, extracting the obtained extract water suspension solution with petroleum ether, collecting the petroleum ether layer, and removing the solvent to obtain the pomelo oil.

The invention has no special restriction on the adding amount of water, and is suitable for fully and uniformly mixing the extract. In the present example, 90g of the extract was dissolved in 800mL of water. The volume ratio of the extract water solution to the petroleum ether is 1 (0.9-1.1), and the preferable ratio is 1: 1. The method of extraction is not particularly limited in the present invention, and any extraction method known in the art may be used. The method for removing the solvent preferably comprises concentration under reduced pressure the method for concentration is not particularly limited, and a concentration scheme well known in the art, such as concentration under reduced pressure, may be employed. The pressure of the reduced pressure concentration is 0.1Mpa, and the temperature of the reduced pressure concentration is preferably 42-47 ℃, and more preferably 45 ℃. The time for the reduced pressure concentration is 3-5 h, and more preferably 4 h.

By adopting the preparation method provided by the invention, the yield of the pomelo oil is more than 4.9% (calculated according to the weight of the dry pomelo seeds). The prepared shaddock oil is measured by a GC-MS method, and the gas chromatography detection conditions are as follows: carrier gas: high purity helium gas; a chromatographic column: HP-5(30.00 m.times.0.25 mm.times.0.25 μm); column temperature: 80 ℃; temperature programming: increasing the temperature from 80 deg.C to 280 deg.C (50min) at a rate of 4 deg.C/min, and maintaining at 280 deg.C for 7min (57 min); no flow diversion; flow rate of carrier gas: 1 mL/min; sample introduction amount: 1 μ L. Mass spectrum detection conditions: an ion source: EI; the mass scanning range is 40-550 amu; ion source temperature: 230 ℃ to 230 ℃. Weighing 2mg of the grapefruit seed PE phase, adding 0.5mL of n-pentane, dissolving in a sample bottle, and determining according to the determination condition setting method of the gas chromatography and the mass spectrum. The detection result shows that the pomelo oil comprises the following active ingredients: d-limonene, (E) -2-decenal, (E, Z) -2, 4-decadienal, (Z, Z) -2, 4-decadienal, tetradecene, beta-caryophyllene, yanogene, hexadecene, (Z, E) -6, 9-heptadecadiene, 8-heptadecene, octadecene, nootkatone, phytone, methyl palmitate, palmitic acid, ethyl palmitate, methyl linoleate, methyl 10-octadecenoate, linoleic acid, ethyl linoleate and ethyl oleate. The pomelo oil preferably comprises the following active ingredients in percentage by mass: 22.86% of D-limonene, 1.18% (E) 2-decenal, 7.77% (E, Z) 2, 4-decadienal, 12.40% (Z, Z) 2, 4-decadienal, 0.64% of tetradecene, 1.47% of beta-caryophyllene, 3.38% of elemene, 0.65% of hexadecene, 1.28% (Z, Z) 6, 9-heptadecadiene, 0.83% of 8-heptadecene, 0.71% of octadecene, 1.18% of nootkatone, 0.71% of phytone, 1.11% of methyl palmitate, 15.79% of palmitic acid, 2.42% of ethyl palmitate, 1.05% of methyl linoleate, 0.52% of 10-octadecenoic acid methyl ester, 20.21% of linoleic acid, 1.88% of ethyl linoleate and 1.97% of oleic acid ethyl ester.

The invention provides a pesticide suitable for cowpea aphids, which takes the pomelo oil or the pomelo oil prepared by the preparation method as an active ingredient. Compared with a blank control group, the low-dose (0.25 mu g/aphid) shaddock oil can achieve the effect of killing the cowpea aphids, and the high-dose (2.5 mu g/aphid) shaddock oil or pesticide has the insecticidal rate of 60 percent when acting for 12 hours and the insecticidal rate of 100 percent when acting for 72 hours continuously.

The formulation of the pesticide is not particularly limited in the present invention, and may be in the form of a pesticide well known in the art, for example, an emulsifier. The emulsifier in the pomelo oil emulsifier comprises Tween 80 or Tween 20. In the embodiment of the invention, the emulsifier is preferably an emulsifier containing tween 80-pomelo oil at a volume concentration of 0.1%. The method for preparing the pesticide is not particularly limited, and the pesticide preparation scheme well known in the field can be adopted.

The invention provides application of the shaddock oil or the pesticide applicable to cowpea aphids in prevention and control of the cowpea aphids. Experiments show that compared with a blank control group, the shaddock oil or the pesticide can obviously improve the insecticidal efficiency after being administered for 12 hours, and the insecticidal effect is the best when the pesticide acts for 72 hours.

The invention provides application of the shaddock oil or the pesticide applicable to the cowpea aphids in reducing the activity of aphid protease. Preferably, the aphid proteases comprise acetylcholinesterase, glutathione S-transferase and peroxidase. Experiments show that the shaddock oil or pesticide has the greatest influence on the activities of acetylcholinesterase, glutathione S-transferase and peroxidase when acting on cowpea aphids for 24 hours, and the change of the enzyme activity is in positive correlation with the dosage.

The following examples are provided to illustrate details of the present invention, a grapefruit oil with insecticidal activity against cowpea aphid, and its insecticide and application, but they should not be construed as limiting the scope of the present invention.

Example 1

Preparation method of pomelo oil

1. Material type and source:

picking Citrus grandis of Citrus of Rutaceae in 2019 s from an orchard of Nankang region of Ganzhou, Jiangxi province, removing its peel and pulp, selecting Citrus grandis seeds, air drying, weighing 502.395g, cutting with scissors, and storing in refrigerator at 4 deg.C.

2. The extraction process of the pomelo oil comprises the following steps:

and (3) extracting the total extract: 250g of chopped pomelo seeds are put into a 1000mL round-bottom flask, 300mL of 95% ethanol water solution is added for soaking overnight, then the heating reflux is carried out for 2h at 80 ℃, the extract is poured out and is concentrated in a clean round-bottom flask under the reduced pressure at 45 ℃ by a rotary evaporator until all ethanol is removed, and the extract is obtained. And adding 300mL of 95% ethanol aqueous solution into the once-refluxed pomelo seeds, refluxing for 2h, concentrating the leachate under reduced pressure to obtain an extract, and performing the same operation on the rest pomelo seeds to obtain 90.7282g of a total extract (namely a crude extract).

And (3) extracting the total extract: pouring the extracted total extract into a 2000mL beaker, adding 800mL of distilled water, and stirring forcefully until the total extract is in a uniform suspension stateAfter conditioning, it was poured into a 2000mL separatory funnel and an equal amount (V) was added_{Aqueous solution}:V_{Petroleum ether}1:1), rapidly oscillating, standing for 6 hours, discharging lower-layer liquid after layering, pouring out an upper-layer petroleum ether organic phase from the upper end, continuously extracting a lower-layer aqueous solution twice with petroleum ether, pouring the upper-layer petroleum ether extract phase into a round bottom flask, and removing the solvent by vacuum concentration with a rotary evaporator to obtain 24.7082g of grapefruit seed oil.

The prepared grapefruit seed oil is measured by a GC-MS method, and the specific contents are as follows:

1) gas chromatography detection conditions

Using an Agilent 7890B gas chromatograph and an Agilent quality detector (Agilent technologies, Santa Clara, Calif., USA); carrier gas: high purity helium gas; a chromatographic column: HP-5(30.00 m.times.0.25 mm.times.0.25 μm); column temperature: increasing the temperature from 80 deg.C to 280 deg.C (50min) at a rate of 4 deg.C per minute, and maintaining at 280 deg.C for 7min (57 min); no flow diversion; flow rate of carrier gas: 1 mL/min; sample introduction amount: 1 μ L.

2) Mass spectrometric detection conditions

An ion source: EI; the scanning range is 40-550; ion source temperature: 230 ℃ to 230 ℃.

2mg of shaddock oil is weighed and added into 0.5mL of n-pentane to be dissolved in a sample bottle, and the measurement is carried out according to the method for setting the measurement conditions of the gas chromatography and the mass spectrum. Then 0.1mL of a mixture of C7-C40 n-alkanes was dissolved in 0.5mL of n-pentane and analyzed by GC-MS equipment under the same conditions as above. Finally, the components of the shaddock oil were identified by NITS (NIST, 2010 edition, department of commerce, gaithersburg, maryland, usa), the actual Retention Index (RI) was calculated using Kovats index (Kovats index) formula I, and analyzed in combination with literature comparisons.

RI ═ 100Z +100[ TR (x) -TR (Z) ]/[ TR (Z +1) -TR (Z) ] formula I

Wherein Z and Z +1 are the numbers of carbon atoms contained in the n-alkanes before and after the efflux of the target compound (X), TR (X) represents the retention time of the target compound X, TR (Z) represents the retention time of the n-alkanes before the efflux of the target compound X, and TR (Z +1) represents the retention time of the n-alkanes after the efflux of the target compound X, respectively.

The GC-MS measurement spectrum is shown in FIG. 1. From GC-MS detection spectra, it can be seen that: the PE phase of the Nankang sweet pomelo seeds contains 21 characteristic obvious peaks, which indicates that the PE phase contains 21 different organic compounds, the structures of the compounds are determined by calculation retention index and literature comparison and NIST-MS retrieval, and the compounds are respectively D-limonene, (E) -2 decenal, (E, Z) -2, 4-decadienal, (Z, Z) -2, 4-decadienal, tetradecene, beta-caryophyllene, eleosteanacene, hexadecene, (Z, E) -6, 9-heptadecadiene, 8-heptadecene, octadecene, nootkatone, phytone, methyl palmitate, palmitic acid, ethyl palmitate, methyl linoleate, methyl 10-octadecenoate, linoleic acid, ethyl linoleate and ethyl oleate. Wherein, the contents of D-limonene, (Z, Z) -2, 4-decadienal, palmitic acid and linoleic acid are higher (the relative content is more than 10 percent), and the contents of other components are relatively less, and the specific information is shown in Table 1.

TABLE 1 active ingredient information of grapefruit oil prepared by the present invention

Example 2

Preparation method of pomelo oil

1. Material type and source:

picking Citrus grandis of Citrus of Rutaceae in 2019 s from an orchard of Nankang region of Ganzhou, Jiangxi province, removing its peel and pulp, selecting Citrus grandis seeds, air drying, weighing 502.395g, cutting with scissors, and storing in refrigerator at 4 deg.C.

2. The extraction process of the pomelo oil comprises the following steps:

and (3) extracting the total extract: placing 30g of chopped pomelo seeds into a 100mL round-bottom flask, adding 35mL of 90% ethanol water solution, soaking overnight, heating and refluxing at 82 ℃ for 2.5h, pouring out the leaching solution, placing in a clean round-bottom flask, performing reduced pressure concentration at 45 ℃ by using a rotary evaporator until all ethanol is extracted, and obtaining extract. And adding 30mL of 90% ethanol aqueous solution into the once-refluxed pomelo seeds, refluxing for 2h, concentrating the leachate under reduced pressure to obtain an extract, and performing the same operation on the rest pomelo seeds to obtain 9.813g of a total extract (namely a crude extract).

And (3) extracting the total extract: pouring the total extract into 200mL beaker, adding 80mL distilled water, stirring to obtain water solution, pouring into 200mL separating funnel, and adding equal amount of (V)_{Aqueous solution}:V_{Petroleum ether}1:1), rapidly oscillating, standing for 7 hours, discharging lower-layer liquid after layering, pouring an upper-layer petroleum ether organic phase from the upper end, continuously extracting a lower-layer aqueous solution twice by using petroleum ether, pouring an upper-layer petroleum ether extraction phase into a round bottom flask, and removing a solvent by using a rotary evaporator through reduced pressure concentration to obtain 2.4812g of grapefruit seed oil. The grapefruit seed oil was subjected to active ingredient measurement by the measurement method of example 1, and the results contained 21 compounds of the same kind as in example 1.

Example 3

Preparation method of cowpea aphid shaddock oil emulsifier

The pomelo oil prepared in the example 1 is prepared into pesticide, 9.9mL of the pomelo oil is firstly absorbed by a liquid-transferring gun into a 20mL glass bottle, 0.1mL of Tween-80 emulsifier is added and then dissolved by ultrasonic (the ultrasonic temperature is 25 ℃, and the time is 10min) to prepare the 1% Tween-80-pomelo oil emulsifier.

Example 4

Determination of contact killing activity of cowpea aphids

The test adopts a micro-dripping method to determine the contact killing activity of the pomelo oil on cowpea aphids (adults collected in vegetable garden at the university of Jiangnan teachers), firstly weighing 50mg of the pomelo oil, dissolving the pomelo oil with 1% Tween-80 and diluting the pomelo oil into different concentration gradients (50mg/mL, 40mg/mL, 20mg/mL, 10mg/mL and 5mg/mL) to prepare an insecticide, dripping 0.05 muL of liquid medicine (1% Tween-80 for a control group) on the breast board of the cowpea aphids by using a micro-dripping instrument, dripping 30 drops of each concentration on the breast board of the cowpea aphids to be tested for 3 times in parallel, transferring the cowpea aphids into a culture dish (filter paper with an inner pad diameter of 12.5cm and adding a small amount of distilled water) with a writing brush after dripping is finished, and placing the cowpea aphids at the temperature of 25 +/-2 ℃, the relative humidity of 70 +/-5% and the illumination time L: d ═ 14 h: feeding in an illumination incubator for 10 h. Observing results at 12h, 24h, 48h and 72h after the pesticide application, using a soft writing brush to lightly touch the cowpea aphid body under a dissecting mirror, regarding immobile persons and persons who cannot normally react as dead, and calculating the corrected death rate according to a formula II.

Mortality (%) - (% dead/test insect number X100 formula II.

The results are shown in FIG. 2. The result shows that the contact killing activity of the pesticide on the cowpea aphids is obvious, and the mortality rate is higher as the pesticide concentration is higher and the action time is longer, the pesticidal effect is better, and the mortality rate reaches more than 80% in 72 hours.

Example 5

Determination of stomach toxicity activity of cowpea aphid

The test adopts a leaf disc method to determine the stomach toxicity activity of the shaddock oil on cowpea aphids (adults), fresh green beans are respectively put into prepared insecticides (50mg/mL, 40mg/mL, 20mg/mL, 10mg/mL and 5mg/mL) to be soaked for 5s, then are taken out, naturally air-dried and put into a culture dish (the diameter is 12.5cm) filled with filter paper (distilled water is used for moisture retention). The cowpea aphids after 12h of starvation were then placed in petri dishes with 30 heads per dish and the experiment was repeated 3 times. With 1% Tween 80-H₂And O is a control group, the control group is treated by the same method, the death condition of the cowpea aphids is recorded after 12h, 24h, 36h, 48h and 72h, and the corrected death rate is calculated according to the formula III.

Mortality (%) < dead number/test number × 100 equation III

The results are shown in FIG. 3. Experimental results show that the effect of PE is obvious compared with that of cowpea aphid, the effect is not obvious when the effect is short at the beginning, the longer the effect is, the aphid can die when the reagent reaches the stomach, and the death rate is increased along with the increase of the reagent concentration. Therefore, the shaddock oil pesticide has a good insecticidal effect on cowpea aphids in a contact killing or stomach poisoning mode.

Example 6

Research on insecticidal mechanism of shaddock oil

1. Preparation of enzyme solution: by micro-dripRespectively treating wingless cowpea aphids (adults) with consistent sizes with liquid medicines with concentrations of LD30(1.10 mug/mL) and LD50(1.75 mug/mL), treating a control group with 1% Tween 80, transferring the treated aphids to a culture dish with fresh green beans by using a writing brush, weighing 11mg of living aphids after 4 hours, 8 hours, 12 hours and 24 hours respectively, putting the aphids into a precooled mortar, and adding NaH₂PO₄﹣Na₂HPO₄The buffer solution (0.01mol/L, pH 7.4) was homogenized in an ice bath, and the homogenate was centrifuged at 4000r/min at 4 ℃ for 10min, and the supernatant was taken as an enzyme solution. The enzyme solution is used for measuring the content of total protein in aphids, the activity of acetylcholinesterase, glutathione-S-transferase and the activity of peroxide.

2. Determination of in vivo protein of cowpea aphid

The total protein content in aphids was determined by the Coomassie Brilliant blue method, by first weighing 0.01g bovine serum albumin and adding 5mL PBS (0.01mol/L, pH 7.4) to dissolve to prepare a stock solution, and then diluting the stock solution with PBS (0.01mol/L, pH 7.4) to different concentrations (1mg/mL, 0.8mg/mL, 0.6mg/mL, 0.4mg/mL, 0.2mg/mL, 0.1mg/mL, 0.05 mg/mL). 270. mu.L of 0.01% Coomassie brilliant blue G-250 and 10. mu.L of bovine serum albumin at different concentrations were added to a 96-well plate, incubated at 25 ℃ for 5min, and immediately thereafter OD was measured at 595nm and 25 ℃ in a microplate reader. And (4) preparing a standard curve according to the concentration and OD value of different bovine serum albumin, and determining the protein content in the aphids by using the same method.

3. Determination of acetylcholinesterase (AchE) activity in cowpea aphid

Preparing substrate acetylthiocholine iodide (AchE) with concentration of 0.02mol/L and color-developing agent 5,5' -dithiobis 2-nitrobenzoic acid (DTNB) with concentration of 0.001mol/L, sequentially adding 50 μ L of enzyme solution, 150 μ L of substrate and 50 μ L of color-developing agent into 96-well plate, immediately measuring with enzyme-labeling instrument at 30 deg.C and 415nm once every 30s for 30min, and totally measuring for 30 min. The control group replaced 50 μ L of the experimental group enzyme with 50 μ L of control enzyme (in vivo enzyme from aphid treated with 1% tween 80), the blank group replaced 50 μ L of enzyme with 50 μ L of 0.04mol/LPBS buffer (pH 8), and the enzyme activity was expressed as the change in OD over 20 minutes, i.e.: the enzyme activity is delta OD/20 min.

4. Determination of glutathione-sulfur-transferase (GST) enzymatic activity in cowpea aphid body

The substrate Glutathione (GSH) concentration is 6mmol/L, and the chromogenic reagent 1-chloro-2, 4-dinitrobenzene (CDNB) concentration is 1.2mmol/L (0.0242 g CDNB is first weighed and dissolved in 1mL absolute ethanol, and then diluted to 1.2mmol/L with 0.1mol/L, pH ═ 7.6 PBS). mu.L of enzyme solution, 100. mu.L of Glutathione (GSH) and 100. mu.L of 1-chloro-2, 4-dinitrobenzene (CDNB) were sequentially added to a 96-well plate, and OD values were rapidly measured with a microplate reader at 30 ℃ and 340nm at 30s intervals for 30min in total. In the control group, 10. mu.L of the control enzyme (in vivo enzyme of aphid treated with 1% Tween 80) was used instead of 10. mu.L of the enzyme in the experimental group, the other reagents were used in the same amounts, and the enzyme activity was expressed by the change in OD value within 20 minutes, namely: the enzyme activity is delta OD/20 min.

5. Activity assay of cowpea aphid Peroxidase (POD)

Add 50mL of NaH to 300. mu.L of analytically pure guaiacol in a beaker₂PO₄﹣Na₂HPO₄The buffer solution (0.05mol/L, pH 6.4) was then placed on a magnetic stirrer and heated until the guaiacol was completely dissolved, 50. mu.L of phosphoric acid buffer solution was added to make 0.3% guaiacol, and 0.3% H₂O₂(dissolved in 0.05mol/L phosphate buffer pH 6.4). 50 μ L of enzyme solution, 150 μ L of 0.3% guaiacol, and 50 μ L of 0.3% hydrogen peroxide (H) were sequentially added to a 96-well plate₂O₂) The OD value was measured by a microplate reader at 30 ℃ and 470nm at intervals of 30 seconds for 30 min. In the control group, 50. mu.L of the control enzyme (in vivo enzyme of aphid treated with 1% Tween 80) was used instead of 50. mu.L of the enzyme of the experimental group, the other reagents were used in the same amounts, and the peroxidase activity was expressed as the change in OD value within 20 minutes, i.e.: the enzyme activity is delta OD/20 min.

The results of the determination of total protein in cowpea aphid are shown in FIG. 4. As can be seen from fig. 4: the total protein content of the cowpea aphid treated by the insecticide in vivo is lower than that of the control group, and the total protein content is reduced along with the increase of the medication concentration, and the total protein content of the experimental group is much lower than that of the control group by 24 hours and the medication concentration is LD₅₀(1.75. mu.g/mouse), the reduction in total protein content was greatest.

The result of the determination of acetylcholinesterase activity in cowpea aphid is shown in figure 5. The acetylcholinesterase activity in the cowpea aphids treated by the insecticide is lower than that in the control group, the enzyme activity in the experimental group is increased firstly and then the change trend is reduced, but the total enzyme activity is still lower than that in the control group. The decrease in acetylcholinesterase activity was greatest in the experimental group by 24h, and the decrease in acetylcholinesterase activity was greater with higher concentrations of the reagents.

The results of measuring the glutathione-S-transferase activity in cowpea aphid are shown in figure 6. As can be seen from FIG. 6, the glutathione S-transferase has reduced enzyme activity as a whole along with the change of time, the time for starting the action is short, the change of the enzyme activity is not obvious, the enzyme activity is reduced remarkably when the action time reaches 24 hours when the concentration of the reagent is 1.75 mug/mL, and the influence of PE on the glutathione S-transferase is great.

The result of peroxidase activity measurement in vivo of Aphis pymetropolis is shown in FIG. 7. As can be seen from FIG. 7, the peroxidase activity gradually decreases with the increase of the action time when the low concentration reagent is acted by 1.10. mu.g/mL with the increase of the action time, and the enzyme activity is reduced to the minimum within 24 h. When the high-concentration reagent is treated by 1.75 mu g/mL, the activity of the peroxidase after 4 hours of action is probably reduced to the minimum, the action time of the high-concentration reagent is short, and the inhibition effect of the enzyme activity is obvious.

The insecticidal mechanism of the pomelo oil prepared by the invention is that the total protein content in the cowpea aphid body is reduced by reducing the activities of acetylcholinesterase, peroxidase and glutathione S-transferase in the cowpea aphid body, so that the insecticidal purpose is achieved.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.