Application of sambucus williamsii extract in preparation of insecticide for plutella xylostella
The diamondback moth insecticide is prepared by elderberry extract, which solves the problem of diamondback moth's resistance to chemical insecticides, achieves environmentally friendly and efficient prevention and control effects, and reduces the negative impact on farmland ecosystems.
Patent Information
- Application Number
- CN202510717885.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-30
- Publication Date
- 2025-09-16
AI Technical Summary
The diamondback moth has developed significant resistance to existing chemical insecticides, resulting in a significant reduction in the effectiveness of traditional chemical control. Natural enemies and pollinators in farmland ecosystems have been adversely affected. There is an urgent need to develop environmentally friendly and efficient control strategies.
The extracts of elderberry stems, leaves and fruits were used to prepare insecticides through ultrasonic extraction and water bath extract solvent extraction. The active ingredients such as neostemonine, 14,15-dehydrogenated ginsenoside, octadecadien-6-ynoic acid, betaine and N-acetylglycine were separated and used to prepare insecticides for Plutella xylostella.
It achieves effective prevention and control of diamondback moth, shows significant insecticidal activity and repellent and antifeedant effects, and reduces the negative impact of the use of chemical pesticides on the environment.
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Figure CN120642856A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of agricultural pest control, and particularly relates to application of an elderberry extract in preparing a diamondback moth insecticide. Background Art
[0002] The diamondback moth, Plutella xylostella (Linnaeus), belongs to the family Plutellidae in the order Lepidoptera. It is the most difficult pest to control on cruciferous vegetables worldwide, currently causing $4-5 billion in losses to the global economy annually. Plutella xylostella has developed significant resistance to over 50 insecticides in three major classes, including organophosphates, carbamates, and pyrethroids, with resistance levels generally reaching moderate to high levels, significantly reducing the effectiveness of traditional chemical control. Against this backdrop, the development of sustainable, environmentally friendly control strategies that can slow the development of insecticide resistance has become an urgent need for integrated agricultural pest management. Existing research indicates that the frequent and long-term application of insecticides in current control systems has led to cross-resistance among target pests to multiple compounds, resulting in frequent control failures in the field and increasing economic losses.
[0003] In agricultural and forestry production practices, effective prevention and control of pest populations that restrict crop yields has become a key technical problem that needs to be solved urgently. Currently, the widespread use of synthetic insecticides has caused many adverse effects on natural enemies and pollinators in farmland ecosystems, such as: extended life cycle, reduced reproductive capacity, changes in predatory behavior, and even death. Botanical insecticides have shown significant development potential due to their high safety, significant efficiency, environmental friendliness, strong targeting, excellent degradation performance, and harmlessness to mammals. Given the diversity of plant resources in nature, many plants with insecticidal activity have not yet been fully developed and studied. Therefore, exploring new, environmentally friendly and efficient pest control strategies has become a top priority. Summary of the Invention
[0004] The present invention aims to provide an effective prevention and control measure for diamondback moth.
[0005] The present invention provides an application of an elderberry extract in preparing an insecticide.
[0006] It is further defined that the insecticide is an insecticide for killing Plutella xylostella.
[0007] It is further defined that the elderberry extract is any one or more of an elderberry stem extract, an elderberry leaf extract, and an elderberry fruit extract.
[0008] It is further defined that the preparation method of elderberry extract is as follows: weigh elderberry dry powder, use 40 times the volume of ethanol solution of the dry powder as solvent, perform ultrasonic extraction for 50 minutes, temperature 45°C, power 360W, let stand, filter the extract, and concentrate and evaporate to obtain an extract.
[0009] It is further defined that the solvent is an ethanol solution having a volume of 40 times that of the elderberry dry powder.
[0010] It is further defined that the ethanol solution is anhydrous ethanol.
[0011] It is further defined that the preparation method of elderberry extract is as follows: the crude ethanol extract of the elderberry plant sample is added with 10 times distilled water to form a suspension, stirred and dissolved at 50°C, placed in a separatory funnel for suspension, and extracted three times with an equal amount of petroleum ether to the solution, the extracts are combined, and the extracts are concentrated under reduced pressure to obtain petroleum ether extract and water extract extracts, respectively.
[0012] It is further defined that the preparation method of the crude ethanol extract of the elderberry plant sample is to obtain the elderberry dry powder by ultrasonic extraction and water bath extract solvent extraction.
[0013] It is further defined that the effective concentration of the extract is 20-100 mg / mL.
[0014] It is further defined that the active ingredients with the highest content in the extract are neostemonine, 14,15-dehydrogenated ginsenoside, octadecadien-6-ynoic acid, betaine and N-acetylglycine.
[0015] Beneficial Effects: The crude and extracts of elderberry were used to test and screen for activity against diamondback moth. The petroleum ether extract, which contained the most active ingredients, was subjected to silica gel column chromatography, preparative thin-layer chromatography, and liquid chromatography-mass spectrometry. The compounds with the highest content, including neostemonine, 14,15-dehydrogenated ginsenoside, octadecadien-6-ynoic acid, betaine, and N-acetylglycine, were isolated. These results provide new green control agents and technologies for the prevention and treatment of diamondback moth. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 is a separation flow chart;
[0017] Figure 2 This is the result of elderberry extract treatment on the symptoms of the third-instar larvae of diamondback moth; DETAILED DESCRIPTION
[0018] Example 1: Preparation of Elderberry Crude Extract and Extracts
[0019] (1) Wash the stems, leaves, and fruits of elderberry, dry them naturally in the shade, and dry them in an electric constant-temperature blast drying oven (Shanghai Senxin Experimental Instrument Co., Ltd.) at 50°C. Then, crush them in a high-speed multifunctional grinder (Shanghai Lijian Machinery Co., Ltd.) and sieve them through 60 mesh to obtain a mixture of stems, leaves, and fruits. The sieved plant sample powder was sealed in a sealed bag and stored in a light-proof cabinet until use.
[0020] (2) The crude elderberry extract was extracted by ultrasonic extraction. 540 g of the elderberry dry powder obtained in step (1) was weighed and divided equally into 22 1,000 mL Erlenmeyer flasks. 40 times the volume of anhydrous ethanol solution of the dry powder was added to each flask as a solvent. The flasks were sealed with plastic wrap, sealing film, and rubber bands and placed in an ultrasonic cleaning machine. The ultrasonic extraction time was 50 min, the temperature was 45 ° C, the power was 360 W, and the extract was allowed to stand for 24 hours. The extract was filtered and transferred to a centrifuge bottle. The extract was concentrated and evaporated using a rotary evaporator (Shanghai Yarong Biochemical Instrument Factory) to obtain 102.88 g of extract. The extract was collected in a brown reagent bottle and stored in a refrigerator at 4 ° C for later use.
[0021] (3) The elderberry extract was extracted by water bath extract solvent extraction. 102.88 g of the extract obtained in step (2) was taken, 10 times the amount of distilled water was added to make a suspension, placed in a 50°C water bath and stirred to dissolve, and placed in a separatory funnel for suspension. The extract was extracted three times using an equal amount of petroleum ether to the solution. The light petroleum ether extract solution was in the upper layer, and the heavy water extract extract was in the lower layer. The extracts were combined, the rotary evaporator speed was set to 100 rpm, the water bath temperature was set to 45°C, and the extracts were concentrated under reduced pressure to obtain 25.00 g of petroleum ether extract and 34.50 g of water extract extract. The petroleum ether extract was collected in a brown reagent bottle and stored in a refrigerator at 4°C for later use.
[0022] Example 2: Separation of Elderberry Active Fraction and Detection of Substance Composition of Active Fraction Fr3-1
[0023] (1) Thin layer chromatography. Based on the development of the chromatography plate in different developing agents, a developing agent with a large number of developing points and an Rf value of about 0.2-0.3 is selected as the eluent for silica gel column chromatography of the fraction. The specific method is as follows:
[0024] Operation process: First, use a glass cutter to cut a 10*20cm silica gel thin layer plate into a 2*5cm chromatography plate. Use a capillary tube to spot the sample solution at a point 0.5cm from the bottom of the chromatography plate. The spot diameter does not exceed 2mm and is the origin. After the solvent of the sample point (the petroleum ether extract obtained in step (3) of Example 1) has completely evaporated, the chromatography plate is placed in a chromatography tank containing a certain amount of developing agent. The sample to be separated is developed. When the solvent has diffused along the chromatography plate to approximately 0.5cm from the top, the chromatography plate is removed and a horizontal line is lightly drawn with a pencil at the position where the solvent has diffused. After the solvent has evaporated, the separated compounds are developed under ultraviolet light at 254nm or 365nm. The distance from the center of the developed spot to the origin is then measured and calculated according to the formula: Rf = distance from the center of the spot to the origin / distance from the solvent front to the origin. The optimal eluent is petroleum ether:methanol.
[0025] (2) Silica gel column chromatography separation. The specific experimental operation process is as follows:
[0026] Column Packing: First, weigh 200-300 mesh silica gel (500g for primary column; 75g for secondary column) into a beaker and add petroleum ether. Stir thoroughly with a glass rod until a homogenous slurry forms, removing any bubbles and setting aside. Slowly pour the silica gel slurry into the chromatography column, ensuring that the silica gel is evenly packed within the column without bubbles or cracks. Allow the column to stand until the height of the silica gel column stabilizes, then wash the column with three column volumes of petroleum ether.
[0027] Sample Addition: (Primary Separation) Weigh 25g of the petroleum ether extract and perform silica gel column chromatography (primary column specifications: 10cm inner diameter, 100cm height). Dissolve the petroleum ether extract in 25mL of petroleum ether. Carefully inject the sample slowly along the column wall with a pipette, then rinse the sample adhering to the column wall with 25mL of petroleum ether. Open the outlet valve and lower the liquid level in the column to the column height, then close the outlet valve. (Fine Separation) Weigh 15g of the petroleum ether extract and perform silica gel column chromatography (secondary column specifications: 3cm inner diameter, 30cm height). Dissolve the petroleum ether extract in 15mL of petroleum ether. Carefully inject the sample slowly along the column wall with a pipette, then rinse the sample adhering to the column wall with 15mL of petroleum ether. Open the outlet valve and lower the liquid level in the column to the column height, then close the outlet valve.
[0028] Elution: Normal-phase silica gel column chromatography was performed using a gradient elution system with increasing polarity. Fractions were quantitatively collected based on the amount of silica gel packed in the column. After TLC analysis, fractions with the same or similar compositions were combined. The primary column was eluted with a gradient of petroleum ether:methanol (20:0, 18:1, 14:1, 12:1, 8:1, 6:1, 14:3, 6:3, 2:4, 0:20, v / v). The elution volume of each gradient elution solution was 1500 mL. 500 mL fractions were collected and numbered sequentially. The secondary column was eluted with a gradient of petroleum ether:methanol (20:0, 18:1, 14:1, 12:1, 8:1, 6:1, 14:3, 6:3, 2:4, 0:20, v / v). The elution volume of each gradient elution solution was 270 mL. 100 mL fractions were collected and numbered sequentially. The specific separation flow chart is as follows Figure 1 As shown; Fr3-1 component was obtained.
[0029] (3) LC-MS detection conditions. ① Metabolite extraction: Take 100 μL of sample (the Fr3-1 component obtained in (2) of Example 2) and place it in an EP tube. Add 400 μL of 80% methanol aqueous solution; freeze in liquid nitrogen for 5 minutes; thaw on ice, vortex for 30 seconds, and sonicate for 6 minutes; centrifuge at 5000 rpm and 4°C for 1 minute, take the supernatant and transfer it to a new centrifuge tube, and lyophilize it into a dry powder; add the corresponding 10% methanol solution according to the sample volume to dissolve it, and inject it into LC-MS for analysis. ②Chromatographic conditions: The chromatographic column conditions were XselectHSS T3, 2.5μm, 2.1×150mm; the mobile phase conditions were A: 0.1% formic acid-water, B: 0.1% formic acid-acetonitrile; the column temperature was 50℃; the flow rate was 0.4mL / min; there were 6 gradients in total (gradient 1: 0min, 98% A, 2%B; gradient 2: 2min, 98% A, 2%B; gradient 3: 15min, 0% A, 100%B; gradient 4: 17min, 0% A, 100%B; gradient 5: 17.1min, 98% A, 2%B; gradient 6: 20min, 98% A, 2%B). ③Mass spectrometry conditions: positive ion mode: Curtain Gas: 35 psi; Collision Gas: Medium; IonSpray Voltage: 5500 V; Temperature: 550°C; Ion Source Gas 1:60; IonSource Gas 2:60.
[0030] Example 3: Determination of insecticidal activity of crude and extracted materials against Plutella xylostella
[0031] (1) Contact killing effect. The micro-drip method was used to carry out bioassay on the third-instar larvae of the diamondback moth. The Shanghai green leaves with the diamondback moth were placed in a culture dish (D = 9 cm) lined with moist filter paper. 1 μL of the agent (the crude extract was the substance obtained in (2) of Example 1, and the extract was the substance obtained in (3) of Example 1) was dripped onto the pronotum of the third-instar larvae of the diamondback moth using a micro-syringe. The dish was sealed with plastic wrap and pierced with an insect pin to ensure air permeability. Twenty third-instar larvae of the diamondback moth were placed in each culture dish. The experiment was repeated three times. The control group was acetone. The dishes were placed in an artificial climate chamber: temperature (26 ± 1) ° C, relative humidity 70% to 75%, and light cycle L:D = 16h:8h. The number of dead insects was counted after 1 day, 2 days, and 3 days of treatment. The death of the diamondback moth was judged as follows: when the antennae (or head) and abdomen were touched with an insect pin, the ones that did not move at all were considered dead. The experimental observation results were all carried out under a microscope.
[0032] The mortality rate was calculated according to the formula: mortality rate (%) = number of dead insects / number of test insects × 100; adjusted mortality rate (%) = (mortality rate of treatment group - mortality rate of control group) / (1 - mortality rate of control group) × 100. The results are shown in Table 1. When the crude extract concentration was 100 mg / mL, the adjusted mortality rate of diamondback moth was 88.33% after 72 hours. After the four solvent extracts were treated with 100 mg / mL for 72 hours, the petroleum ether phase had a higher contact activity against diamondback moth, with an adjusted mortality rate of 58.33%. After the petroleum ether extract was treated with a concentration of 200 mg / mL for 72 hours, the adjusted mortality rate of diamondback moth larvae was as high as 93.33%.
[0033] Table 1
[0034]
[0035]
[0036] (2) Repellent effect. The poison-loaded leaf method was used to determine the repellent effect of five concentrations of elderberry crude extract on diamondback moth. Fresh Shanghai green leaves were punched into small discs using a 20 mm diameter puncher. 10 μL of crude extract (the substance obtained in (2) of Example 1) and the control (acetone) were applied to the front and back of the leaves, respectively. After natural drying, two leaves with crude extract and two control leaves were placed crosswise on the front and back of a culture dish lined with moist filter paper (D = 9 cm). 20 3rd-instar diamondback moth larvae were placed in the middle of each culture dish, sealed with plastic wrap and pierced with an insect pin. Each treatment was repeated 3 times. The distribution of the larvae was checked 2h and 4h after treatment and the repellent rate was calculated.
[0037] According to the formula: repellency rate (%) = (number of insects in the control group - number of insects in the test group) / number of insects in the control group × 100. As shown in Table 2, after 4 hours of treatment with the crude extract on the diamondback moth larvae, the repellency level of the group treated with the concentration of 40 mg / mL was V.
[0038] Table 2
[0039]
[0040] (2) Antifeedant effect. The leaf toxicity method was used to determine the selective antifeedant effect of five concentrations of elderberry crude extract (the substance obtained in (2) of Example 1) on diamondback moth larvae. Use a ruler to cut the leaves into 1×1 cm square leaves, and apply the diluted crude extract and acetone control solution respectively. After the square leaves are naturally dried, two pieces each from the control group and the experimental group are placed crosswise and equidistantly in a culture dish lined with moist filter paper (D=9 cm). Three third-instar diamondback moth larvae (starved for 3 hours) are placed in the culture dish, and each treatment is repeated 3 times.
[0041] According to the formula: Antifeedant rate (%) = (Control group feeding area - Experimental group feeding area) / (Control group feeding area + Experimental group feeding area) × 100. As shown in Table 3, when the treatment concentration was 60 mg / mL, the 24-hour antifeedant rate was 100%. This result shows that the elderberry crude extract has a significant antifeedant effect on Plutella xylostella and is suitable for use as an antifeedant agent.
[0042] Table 3
[0043]
[0044] (4) Observation of poisoning symptoms. Symptoms of the third-instar larvae of the diamondback moth treated with elderberry extract are shown in Figure 2 After 24 hours, the diamondback moth test insects died, lying on their sides with their bodies curled up. They showed symptoms such as rotting, swelling, blackening of the head and abdomen, stiffness, and obstructed molting. (ac refers to contact killing with crude extracts; df refers to contact killing with petroleum ether extracts)
[0045] Experimental Example 4: Determination of the activity of elderberry active fractions against diamondback moth
[0046] The activity test in Example 3 confirmed that petroleum ether has insecticidal activity against Plutella xylostella. Therefore, the following experiments used thin layer chromatography and column chromatography methods, with Plutella xylostella as the test insect, and separated the petroleum ether extract phase using the activity tracking method. According to the steps in (2) of Example 2, 25.00 g of the petroleum ether extract was subjected to silica gel column chromatography for preliminary separation. The Fr3 fraction with the most prominent contact activity against Plutella xylostella was selected for further column chromatography separation to obtain the elderberry insecticidal active ingredient with higher purity.
[0047] The distillate was prepared into a 100 mg / mL solution, and fractions Fr1 to Fr4 were used for activity testing against Plutella xylostella. Table 4 shows that among the primary silica gel column fractions of the petroleum ether extract, fraction Fr3 exhibited the strongest insecticidal activity, achieving a mortality rate of 76.67% against Plutella xylostella larvae. Among the secondary fractions, fraction Fr3-1 achieved the highest mortality rate against Plutella xylostella larvae, achieving a mortality rate of 65.00%.
[0048] Table 4
[0049]
[0050] Experimental Example 5: Qualitative Analysis of Material Composition of Fraction Fr3-1
[0051] The results of Example 4 show that the primary fraction Fr3 has good contact activity against Plutella xylostella, and the secondary fraction Fr3-1 has the best contact activity against Plutella xylostella. The components of the secondary fraction Fr3-1 will be analyzed below.
[0052] LC-MS analysis was performed according to the method in (3) of Example 2. The mass spectrometric data of the components obtained from the analysis of fraction Fr3-1 were retrieved from the KEGG, HMDB, and LIPIDMaps libraries and compared with relevant standard chromatographic data. The relative content of each component was determined by peak area normalization.
[0053] Thirty-eight components were identified from fraction Fr3-1 (Table 5), accounting for 92.01% of the total composition. The results indicate that fraction Fr3-1 exhibits a complex chemical composition, primarily consisting of amino acids and their derivatives, alkaloids and their derivatives, lipids, and terpenoids. Among these components, the highest concentrations included neotuberostemonine (9.576%), 14,15-dehydrocrepenynic acid (8.059%), octadecadien-6-ynoic acid (7.936%), betaine (4.854%), and N-acetylglycine (4.337%). Therefore, these key components may be responsible for the potent insecticidal activity of elderberry against Plutella xylostella.
[0054] Table 5
[0055]
[0056]
Claims
1. Application of elderberry extract in the preparation of insecticides.
2. The use according to claim 1, characterized in that The insecticide is a diamondback moth insecticide.
3. The use according to claim 1, characterized in that The elderberry extract is any one or more of an elderberry stem extract, an elderberry leaf extract, and an elderberry fruit extract.
4. The use according to claim 1, characterized in that The preparation method of elderberry extract is as follows: weigh elderberry dry powder, use 40 times the volume of ethanol solution of the dry powder as solvent, perform ultrasonic extraction for 50 minutes, temperature 45°C, power 360W, let it stand, filter the extract, and concentrate and evaporate to obtain an extract.
5. The use according to claim 4, characterized in that An ethanol solution of 40 times the volume of elderberry dry powder was used as the solvent.
6. The use according to claim 4, characterized in that The ethanol solution is anhydrous ethanol.
7. The use according to claim 1, characterized in that The preparation method of elderberry extract is as follows: the crude ethanol extract of the elderberry plant sample is added with 10 times distilled water to form a suspension, the solution is dissolved by stirring at 50°C, and the solution is placed in a separatory funnel for suspension. The extracts are extracted three times using an amount of petroleum ether equal to that of the solution. The extracts are combined and concentrated under reduced pressure to obtain petroleum ether extract and water extract extracts, respectively.
8. The use according to claim 7, characterized in that The preparation method of the crude ethanol extract of the elderberry plant sample is to obtain the elderberry dry powder by using an ultrasonic extraction method and a water bath extract solvent extraction method.
9. The use according to claim 1, characterized in that The effective concentration of the extract is 20-100 mg / mL.
10. The use according to claim 1, characterized in that The active ingredients with the highest content in the extract are neostemonine, 14,15-dehydrogenated ginsenoside, octadecadiene-6-ynoic acid, betaine and N-acetylglycine.