Spiro sesquiterpene compound as well as preparation method and application thereof

Abstract

The invention relates to a spiro sesquiterpene compound as well as a preparation method and application thereof. The spiro sesquiterpene compound comprises a compound with a structure as shown in a formula (1). The compound provided by the invention is extracted from dry roots of a cultivated plant eggplant, can be used for preparing a pesticide, is safe to human and livestock, has remarkable activity on agricultural, warehousing and sanitary pests, and has a wide insecticidal spectrum, and the pests are not easy to generate drug resistance after being used. Formula (1)

Description

Technical Field

[0001] This invention belongs to the field of plant-derived pesticide technology, and particularly relates to a spirocyclic sesquiterpene compound, its preparation method, and its application. Background Technology

[0002] In modern agricultural pest and disease control systems, chemical pesticides still occupy a crucial position. However, their drawbacks, such as high application rates, easy induction of pesticide resistance in pests and diseases, and significant negative ecological impacts, are increasingly becoming a bottleneck restricting the high-quality development of the pesticide industry and crop cultivation. Plant secondary metabolites, as an important class of bioactive substances, can provide highly active lead structures for the design and discovery of innovative pesticide molecules, and can themselves be used to create eco-friendly plant-derived pesticides. This control strategy based on natural products can not only effectively manage pests and diseases, but also significantly reduce the risk of pesticide residues and environmental pollution caused by chemical pesticides, which is of great value in promoting the green and sustainable development of agriculture. Therefore, screening for natural compounds with insecticidal properties will be of great significance for the development of green pesticides. However, obtaining plant secondary metabolites with effective insecticidal activity remains a technical challenge.

[0003] Eggplant (Solanum melongena) is an important vegetable crop, but there are currently no reports of eggplant-derived spirocyclic sesquiterpenes that can be used for insecticidal purposes, and there are no reports of using eggplant-derived spirocyclic sesquiterpenes to control pests in agriculture, storage, or sanitation. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present invention provides a spirocyclic sesquiterpene compound, its preparation method and application. This compound is extracted from the dried root of the cultivated plant eggplant (Solanum melongena). This compound does not contain chemical pesticide components, is safe for humans and animals, and pests are unlikely to develop resistance after use. It exhibits significant activity against agricultural, storage, and sanitary pests, and has a broad insecticidal spectrum.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: This invention provides a spirocyclic sesquiterpene compound, comprising compounds with the structure shown in formula (1). Equation (1).

[0006] The beneficial effects of adopting the above technical solution include: This invention extracts the aforementioned spirocyclic sesquiterpene compounds from the dried roots of eggplant, which exhibit good insecticidal activity and a broad insecticidal spectrum, possessing significant pesticide application value. It can be used to control agricultural, storage, and sanitary pests with remarkable effects. Furthermore, since this compound is derived from the edible plant eggplant, it is safe for humans. Compared to synthetic pesticides, this compound is easily degraded in the environment, exhibiting safe and low-residue characteristics. By reducing the frequency and dosage of existing chemical pesticides, it contributes to the green development of protected areas and the sustainable cultivation of high-value-added crops in my country.

[0007] This invention provides a method for preparing the above-mentioned spirocyclic sesquiterpene compound, comprising the following steps: (1) Using eggplant root as raw material, ethanol extraction was performed to obtain ethanol extract; (2) The ethanol extract was processed by silica gel column chromatography, and gradient elution was performed using hexane-acetone as the eluent. The fraction obtained by elution with a volume ratio of hexane to acetone of 10:1 was collected. (3) The fractions eluted by hexane and acetone collected in step (2) were processed by silica gel column chromatography with petroleum ether-acetone as the eluent and the volume ratio of petroleum ether to acetone being 8:1. The eluted fractions of the 4th to 5th column volumes were collected. (4) The petroleum ether and acetone eluent fractions collected in step (3) were purified by gel chromatography using a chloroform-methanol system to obtain spirocyclic sesquiterpenoid compounds.

[0008] The beneficial effects of adopting the above technical solutions include: This invention mines insecticidal components from eggplant, providing lead compounds for the creation of environmentally friendly plant pesticides, effectively reducing the use of chemical pesticides, alleviating the problems of pesticide resistance and pesticide residues in pests and diseases, and ensuring the quality and safety of agricultural products. This invention utilizes eggplant root extracts of active ingredients, realizing the high-value utilization of waste and extending the industrial chain of solanaceous crops. This invention helps to build a sustainable pest control system that combines crop self-defense mechanisms with integrated pest management, which is of great strategic significance for promoting green agricultural development and ecological civilization construction. Research has found that the spirocyclic sesquiterpenoid compounds provided by this invention have significant insecticidal activity against agricultural, storage, and sanitary pests, with novel structures and good environmental compatibility. Their broad-spectrum insecticidal properties provide lead compounds for the development of green pesticides in multiple fields, helping to reduce dependence on chemical pesticides, promoting the green transformation of agricultural, grain storage, and public health pest control, and are of great significance for ensuring food safety and human health.

[0009] Furthermore, in step (1), the alcohol extraction is performed 2-4 times, the temperature of each alcohol extraction is 25-30℃, and the time of each alcohol extraction is 3-7 days.

[0010] Furthermore, in step (2), elution is performed in the order of volume ratio of hexane to acetone in the hexane-acetone system being 20:1, 10:1, 5:1, and 3:1.

[0011] Furthermore, in step (4), purification is performed using Sephadex LH-20 gel.

[0012] Furthermore, in step (4), the volume ratio of chloroform to methanol in the chloroform-methanol system is 1:0.8.

[0013] Furthermore, step (4) also includes a step of collecting the eluent from the first and second column volumes and concentrating it to dryness.

[0014] The beneficial effects of adopting the above scheme include: the above method is conducive to obtaining spirocyclic sesquiterpenoid compounds with the structure shown in formula (1). This compound has the advantages of novel structure, good environmental compatibility, broad insecticidal spectrum, good control effect, safety to humans and animals, and low likelihood of developing drug resistance.

[0015] The present invention provides a pesticide comprising the above-mentioned spirocyclic sesquiterpene compound.

[0016] In addition to the above-mentioned spirocyclic sesquiterpenoid compounds, other ingredients commonly used in the art for preparing pesticides can also be added. The present invention does not impose any special restrictions on the formulation of pesticides, and can be the formulations commonly used in the art, such as liquid formulations, solid formulations, sustained-release formulations, and other formulations.

[0017] The beneficial effects of adopting the above-mentioned scheme include: the pesticide provided by the present invention has the advantages of novel structure, good environmental compatibility, broad insecticidal spectrum, good control effect, safety to humans and animals, and low likelihood of developing drug resistance in the prevention and control of agricultural pests, stored pests and sanitary pests.

[0018] This invention provides the application of the above-mentioned spirocyclic sesquiterpene compounds in the preparation of pesticides.

[0019] This invention provides the application of the above-mentioned spirocyclic sesquiterpene compounds or the above-mentioned pesticides in the control of pests.

[0020] The pests may be agricultural pests, storage pests, or sanitary pests. Furthermore, the pests are selected from one or more of the following: aphids, mites, root-knot nematodes, red flour beetles, corn weevils, mosquitoes, and flies.

[0021] This invention provides the above-mentioned spirocyclic sesquiterpene compounds, which exhibit excellent insecticidal activity against agricultural pests (such as aphids, mites, and root-knot nematodes), storage pests (such as red flour beetles and corn weevils), and sanitary pests (such as mosquitoes and flies), and can be used to prepare plant-derived insecticides. The advantages of this invention include: (1) Novel structure and unique origin. The natural spirocyclic sesquiterpene skeleton discovered for the first time in eggplant is significantly different from the molecular structure of existing pesticides. It has independent intellectual property rights and provides a brand-new lead structure for the creation of original green pesticides.

[0022] (2) Broad insecticidal spectrum and excellent activity: A single compound exhibits highly efficient insecticidal activity against three major categories of pests: agricultural (aphids, mites, root-knot nematodes), stored (red flour beetle, corn weevil) and sanitary (mosquitoes, flies), breaking through the limitations of the scope of action of traditional pesticides and possessing the development potential of "one agent for multiple uses".

[0023] (3) Good environmental compatibility: It originates from plant secondary metabolism, is easily degraded in the environment, has low residue and good safety, is safe for non-target organisms, conforms to the green plant protection and sustainable development strategy, and can significantly reduce the ecological risks brought by chemical pesticides.

[0024] (4) It is not easy to develop resistance: the multi-target mechanism of natural products can effectively delay the development of pesticide resistance in pests, solve the resistance problem caused by the single target of traditional chemical pesticides, and extend the product life cycle.

[0025] (5) Renewable resources and controllable costs: Eggplant is widely planted and its resources are sustainable. It can be obtained through extraction or biosynthesis. Compared with fully synthetic pesticides, it has a cost advantage and realizes the high-value utilization of agricultural products, resulting in significant economic and social benefits. Attached Figure Description

[0026] Figure 1 The compound shown in formula (1) (i.e., Solamelin A) 1 H NMR spectrum.

[0027] Figure 2 The compound shown in formula (1) (i.e., Solamelin A) 13 C10 NMR spectrum.

[0028] Figure 3 The image shows the HSQC nuclear magnetic resonance spectrum of the compound (i.e., Solamelin A) shown in formula (1).

[0029] Figure 4 The nuclear magnetic resonance HMBC spectrum of the compound shown in formula (1) (i.e., Solamelin A) is shown.

[0030] Figure 5 The HRESIMS mass spectrum of the compound shown in formula (1) (i.e., Solamelin A) is shown. Detailed Implementation

[0031] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0032] This invention provides a spirocyclic sesquiterpene compound, its preparation method, and its application, including compounds with the structure shown in formula (1). Equation (1).

[0033] The above compound can also be named Solamelin A. The preparation method of the above compound may include the following steps: (1) The dried eggplant roots were extracted with 95% industrial ethanol to obtain an ethanol extract; (2) The ethanol extract was processed by silica gel column chromatography and gradient elution was performed with n-hexane-acetone as the eluent to obtain the Fr2 fraction; (3) The Fr2 fraction was treated by silica gel column chromatography and eluted with petroleum ether-acetone to obtain the Fr2B fraction; (4) The Fr2B fraction was purified by gel column chromatography using a chloroform-methanol system to obtain spirocyclic sesquiterpenoid compounds.

[0034] Specifically, the preparation method of the above-mentioned compound may include the following steps: (1) Take dried eggplant roots, crush them, extract them with 95% ethanol, combine the extracts and concentrate them under reduced pressure to obtain ethanol extract; (2) Take the ethanol extract obtained in step (1) and process it by silica gel column chromatography. The silica gel can be domestically produced column chromatography silica gel with a particle size of 200-400 mesh. Use hexane-acetone as the eluent for gradient elution. The volume ratio of hexane to acetone in the hexane-acetone system is 20:1, 10:1, 5:1, and 3:1, respectively. Collect the eluent and concentrate it under reduced pressure at a vacuum of -0.08 to -0.095 MPa and a temperature of 45-55℃ to obtain four fractions Fr1-Fr4. Among them, the Fr2 fraction is the eluent obtained by elution with petroleum ether-acetone at a volume ratio of 10:1 and then concentrated under reduced pressure. (3) Take the Fr2 fraction and process it by silica gel column chromatography. The silica gel can be domestically produced column chromatography silica gel with a particle size of 200-400 mesh. Use petroleum ether-acetone as the eluent (petroleum ether to acetone volume ratio of 8:1) for elution. Detect by thin-layer chromatography, develop color, combine the same eluted fractions, and concentrate to dryness under reduced pressure to obtain three fractions, namely Fr2A, Fr2B and Fr2C. Among them, the Fr2A fraction is the eluted fraction of the 2nd-3rd column volume, which is combined and concentrated to dryness under reduced pressure. The Fr2B fraction is the eluted fraction of the 4th-5th column volume, which is combined and concentrated to dryness under reduced pressure. The Fr2C fraction is the eluted fraction of the 6th-7th column volume, which is combined and concentrated to dryness under reduced pressure. (4) The Fr2B fraction in step (3) was purified by gel column chromatography and eluted with a chloroform-methanol system with a volume ratio of chloroform to methanol of 1:0.8 to obtain the compound shown in formula (1), namely compound Solamelin A.

[0035] This invention provides the compound Solamelin A for the first time and discovers its wide application in pesticides and other fields. It exhibits broad-spectrum insecticidal effects against aphids, mites, root-knot nematodes, red flour beetles, corn weevils, mosquitoes, and flies. Furthermore, it possesses advantages such as high insecticidal efficacy, high environmental and ecological safety, significant value in resistance management, and excellent economic efficiency.

[0036] Unless otherwise specified, all techniques or conditions used in the examples are conventional methods or performed according to techniques or conditions described in the literature in this field, or according to product instructions. Reagents used, unless otherwise specified, are all conventional products that can be purchased from legitimate channels or prepared according to conventional methods in this field. Instruments used, unless otherwise specified, are all conventional products that can be purchased from legitimate channels.

[0037] The following provides a further detailed description of the invention. However, this should not be construed as limiting the scope of protection of the invention to the following embodiments. All technologies implemented based on the content of this invention fall within the scope of protection of this invention. Based on the content of this invention, and according to ordinary technical knowledge and common practices in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical concept of this invention, and the modified, substituted, or altered solutions are also within the scope of protection of this invention. Example 1

[0038] The preparation of compound Solamelin A includes the following steps: (1) Extraction: Take dried eggplant roots, crush them to a particle size of 20-60 mesh, and extract with 95% ethanol 2-4 times. During each extraction, the mass-to-volume ratio of crushed dried eggplant roots to 95% ethanol is 1 g:5 mL to 1 g:15 mL. Soak the extracts at 25-30℃ for 3-7 days each time. Combine the extracts and concentrate them under reduced pressure at a vacuum of -0.08~-0.095 MPa and a temperature of 45-55℃ until there is no alcohol odor, to obtain an ethanol extract.

[0039] (2) The ethanol extract obtained in step (1) is mixed with an equal mass of silica gel (100-200 mesh) by dry mixing and used as the sample for loading. Silica gel column chromatography is used. The silica gel can be column chromatography silica gel with a particle size of 200-400 mesh and a column diameter-to-height ratio controlled at 1:8 to 1:10. The sample loading amount to silica gel mass ratio is 1:30 to 1:50 (w / w). Commercially available products such as Qingdao Ocean Chemical Plant Column Chromatography Silica Gel (China) can be used. Equivalent silica gels can achieve the same separation effect. Gradient elution is performed using hexane-acetone as the eluent. Elution is performed in the order of hexane-acetone volume ratio in the hexane-acetone system as 20:1, 10:1, 5:1, and 3:1, with each gradient elution consisting of 3 column volumes. Elution with a hexane to acetone volume ratio of 20:1 can elute waxes and pigments with low polarity; elution with a hexane to acetone volume ratio of 5:1 and 3:1 can elute sugars and phenolic acids with higher polarity; elution with a hexane to acetone volume ratio of 10:1 can accurately elute the moderately polar Solamelin A. The eluent obtained by elution with a hexane to acetone volume ratio of 10:1 was collected and concentrated under reduced pressure at a vacuum degree of -0.08 to -0.095 MPa and a temperature of 45-55℃ until there was no alcohol odor (a small amount of residual solvent remained in the form of a paste), while retaining the fluidity for loading in the following step (3), to obtain the Fr2 fraction.

[0040] (3) Take the Fr2 fraction and mix it with an equal mass of silica gel (100-200 mesh) by dry mixing, and use it as the sample for loading. Silica gel column chromatography is used. The silica gel can be domestically produced column chromatography silica gel with a particle size of 200-400 mesh. Eluent is petroleum ether-acetone, with an eluent volume of 7 column volumes and a petroleum ether to acetone volume ratio of 8:1. One tube is collected for every 0.2 column volumes, and detected by thin-layer chromatography. Color development is performed, and the same eluting fractions are combined. Thin-layer chromatography detection uses silica gel GF254 thin-layer plates (coating thickness 0.2 mm, containing fluorescent indicator, Qingdao Ocean Chemical or other equivalent products), which are activated at 110℃ for 30 min before use. The sample loading volume is 5-10 μL (1 mg / mL solution), and the developing solvent is petroleum ether-acetone (8:1, v / v). After drying, the fluorescent spots are observed under a UV lamp (254 nm), or the colorimetric reagent is sprayed and heated for color development.

[0041] Three fractions were obtained using the above method: Fr2A, Fr2B, and Fr2C. The Fr2A fraction, eluted from the 2nd and 3rd column volumes, was combined and concentrated to dryness under reduced pressure. The Fr2B fraction, eluted from the 4th and 5th column volumes, was combined and concentrated to dryness under reduced pressure. The Fr2C fraction, eluted from the 6th and 7th column volumes, was combined and concentrated to dryness under reduced pressure. The reduced pressure concentration was performed at 45°C and a vacuum of -0.09 MPa.

[0042] (4) The Fr2B fraction was purified by gel column chromatography: Sephadex LH-20 gel (120 g, 2.5 cm × 100 cm glass column) was used, and chloroform-methanol (chloroform:methanol = 1:0.8, v / v) was used as the eluent. The eluent volume was 6 column volumes, the flow rate was 0.3-0.5 mL / min, and one tube was collected every 0.2 column volumes. The developing solvent was n-hexane-acetone, with a volume ratio of n-hexane to acetone of 10:1. The product was developed with 10% (v / v) sulfuric acid ethanol. The eluent from the first and second column volumes was combined and concentrated to dryness under reduced pressure at 40 °C and -0.09 MPa. The product was named Solamelin A. Example 2

[0043] After isolating compound Solamelin A, its physicochemical properties and molecular structure were identified, and the spectral results are as follows: Figures 1 to 5 As shown, where, Figure 1 The 1H NMR spectrum of compound Solamelin A. Figure 2 This is the carbon NMR spectrum of the compound Solamelin A. Figure 3 This is the two-dimensional C-H correlation NMR spectrum of the compound Solamelin A. Figure 4 The two-dimensional C-H long-range NMR spectrum of compound Solamelin A. Figure 5 The high-resolution mass spectrum of compound Solamelin A is shown below. Specific physicochemical properties and spectral data are as follows: Solamelin A: Yellow amorphous solid; white powder, HR-ESI-MS m / z 243.1721[M+Na] + (Theoretical calculated value 243.1725, C) 15 H 24 ONa + One-dimensional nuclear magnetic resonance data: 1 H NMR δ 5.74 (dd, J = 9.4,5.2 Hz, 1H), 5.57 (d, J = 5.1 Hz, 1H), 5.54 (dd, J = 9.2, 5.0 Hz, 1H), 2.19 (dd, J = 12.4, 6.1 Hz, 1H), 2.01 (dd, J = 12.1, 7.0 Hz, 1H), 1.75 (s, 3H), 1.40 (t, J = 12.6 Hz, 1H), 1.21 (s, 6H), 0.93 (d, J = 7.2 Hz, 3H). 13C NMR δ141.41, 130.31, 122.72, 119.12, 72.15, 49.36, 49.30, 35.56, 32.60, 31.14,28.36, 27.99, 25.16, 19.80, 13.83. Solamelin A was identified as a spirocyclic sesquiterpene compound, and its structural formula is shown in formula (1).

[0044] Equation (1).

[0045] Example 3: Aphid-killing activity test determination Following the standard insecticidal test methods of the Ministry of Agriculture and Rural Affairs of China, the insecticidal activity of compound Solamelin A against the peach aphid (Myzus persicae) was tested using the leaf immersion method. The specific procedures are as follows: (1) Preparation of test solution: Accurately weigh 10 mg of Solamelin A, dissolve it in 1 mL of acetone to prepare a 10 mg / mL stock solution. Dilute with 0.5% (v / v) Tween-80 sterile aqueous solution to 5 concentrations: 20 mg / L, 10 mg / L, 5 mg / L, 2.5 mg / L, and 1.25 mg / L, with a final acetone concentration ≤1%.

[0046] (2) Leaf disc treatment: Fresh cabbage leaf discs (5 cm in diameter) were immersed in the test drug solution for 10 seconds and then air-dried at 25°C for 30 minutes. Ten leaf discs were treated for each concentration.

[0047] (3) Inoculation: Inoculate 20 wingless adult aphids that are 1-2 days old into each leaf disc, and keep them moist by making holes in plastic wrap.

[0048] (4) Positive control: Prepare solutions of 98% matrine technical material with concentrations of 100 mg / L, 50 mg / L, 25 mg / L, 12.5 mg / L and 6.25 mg / L respectively according to step (1). Treat the leaf discs respectively according to step (2), and perform the remaining operations as with the test drug solution.

[0049] (5) Blank control: an aqueous solution containing acetone and Tween-80 (1% by volume of acetone and 0.5% by volume of Tween-80). The leaf disc was treated according to step (2), and the rest of the operation was the same as the test solution.

[0050] (6) Cultivation: Each group was placed in an artificial climate chamber for cultivation under the following conditions: 25±1℃, RH75%, and photoperiod 16 L:8D.

[0051] (7) Investigation: Microscopic examination after 24 hours; those with movable antennae or legs are considered live insects. The mortality rate of the blank control is ≤5% and is considered valid.

[0052] (8) Statistical analysis: Each treatment was repeated 4 times, the adjusted mortality rate was calculated, and the LD was determined by Probit analysis using SPSS. 50 value.

[0053] The 24-hour mortality rate of the blank control was 3.2% (≤5%, indicating a valid experiment). Solamelin A showed an LD50 result against the peach aphid. 50 The LD50 of matrine was 11.45 mg / L (95% confidence limit 8.72-14.38 mg / L). 50 The effective concentration was 33.62 mg / L (95% confidence limit 26.51-42.15 mg / L), with a relative toxicity index of 2.94. This indicates that the compound Solamelin A provided by this invention exhibits significant insecticidal activity against peach aphids, exceeding that of the positive control.

[0054] Example 4: Determination of acaricide activity Following the standard insecticidal test methods of the Ministry of Agriculture and Rural Affairs of China, the slide immersion method was used to test the insecticidal activity of compound Solamelin A against the carmine spider mite (Tetranychus cinnabarinus). The specific procedures are as follows: (1) Preparation of test solution: Accurately weigh 10 mg of Solamelin A, dissolve it in 1 mL of acetone to prepare a 10 mg / mL stock solution. Dilute with sterile aqueous solution containing 0.5% (v / v) Tween-80, and set 5 concentration gradients, namely 40 mg / L, 20 mg / L, 10 mg / L, 5 mg / L, and 2.5 mg / L (effective concentration), with a final acetone content ≤1% (v / v).

[0055] (2) Test mites: Female adult Tetranychus cinnabarinus were inoculated onto broad bean leaves and kept at 25±1℃, RH 75%, 16 L:8 D. Female adult mites with the same oviposition period were selected for testing.

[0056] (3) Adhesive mites: Apply double-sided tape (12 mm × 12 mm, 0.1 mm thick) to the center of a glass slide (76 mm × 26 mm), and attach 30 female adult mites to each slide, with their backs facing down. Place the slide at 25 ± 1℃ and RH 85% for 4 hours to acclimate, and remove any dead or unhealthy individuals.

[0057] (4) Immersion: Immerse the glass slide with spider mites completely in the test solution for 5 seconds to ensure that all spider mites are wetted. After removal, quickly absorb the excess solution with filter paper (within 2 seconds) to prevent the solution from running away.

[0058] (5) Blank control: an aqueous solution containing acetone and Tween-80 (1% by volume of acetone and 0.5% by volume of Tween-80). The spider mites were treated with this solution in step (4), and the rest of the operation was the same as the test solution.

[0059] (6) Positive control: The 240 g / L spirodiclofen suspension was diluted to 200 mg / L, 100 mg / L, 50 mg / L, 25 mg / L and 12.5 mg / L solutions in the same way as in step (1), and the spider mites were treated with the drug in step (4). The rest of the operation was the same as the test drug solution.

[0060] (7) Incubation: Place the slides of each group in a biochemical incubator (25±1℃, RH 85%, incubation in the dark), and repeat each treatment 3 times.

[0061] (8) Investigation: Microscopic examination was performed 24 hours later. The mites were gently touched with a small brush. Those that did not move at all were considered dead, while those that showed slight tremors in their limbs were considered alive. Corrected mortality rate = (treatment mortality rate - blank control mortality rate) / (1 - blank control mortality rate) × 100%.

[0062] (9) Statistics: LD was calculated using the Probit analysis method in SPSS software. 50 Value and 95% confidence limit.

[0063] The 24-hour mortality rate in the blank control group was 2.8% (≤5%, indicating a valid experiment). Solamelin A's LD50 against the carmine spider mite... 50 The value was 16.98 mg / L (95% confidence limit: 13.24-21.05 mg / L), spirodiclofen LD50. 50 The effective value was 121.58 mg / L (95% confidence limit: 98.32-148.76 mg / L), with a relative toxicity index of 7.16. This indicates that the compound Solamelin A provided by this invention exhibits significant insecticidal activity against the spider mite, far exceeding that of the positive control.

[0064] Example 5: Determination of Root-knot Nematode Activity Following the standard insecticidal test methods of the Ministry of Agriculture and Rural Affairs of China, the insecticidal activity of compound Solamelin A against the southern root-knot nematode (Meloidogyne incognita) was determined using the immersion method. The specific procedures are as follows: (1) Preparation of test solution: Accurately weigh 10 mg of Solamelin A, dissolve it in 1 mL of acetone to prepare a 10 mg / mL stock solution. Dilute with 0.5% (v / v) Tween-80 sterile aqueous solution to set 5 concentration gradients, namely 40 mg / L, 20 mg / L, 10 mg / L, 5 mg / L and 2.5 mg / L, with a final acetone content ≤1% (v / v).

[0065] (2) Preparation of nematode suspension: Second instar larvae (J2) of southern root-knot nematode (Meloidogyne incognita) were incubated in sterile water at 25℃ for 48 h and the concentration was adjusted to 500 nematodes / mL.

[0066] (3) Experimental system: In a 24-well cell culture plate, add 40 μL of nematode suspension (20 nematodes) + 160 μL of test drug solution to each well, with a final volume of 200 μL, and mix gently. Repeat each treatment in 3 wells.

[0067] (4) Blank control: a sterile aqueous solution containing acetone and Tween-80 (1% by volume of acetone and 0.5% by volume of Tween-80). The blank control solution was added to the system in step (3), and the rest of the operation was the same as the test solution.

[0068] (5) Incubation: Each group was placed in an artificial climate chamber (25±1℃, RH 85%, dark incubation) for 48 h.

[0069] (6) Investigation: Microscopic examination was conducted 48 hours later. The insects were gently touched with a small brush. Those that were stiff and inactive were considered dead. The mortality rate of the blank control group was ≤10% to be considered a valid test.

[0070] (7) Statistics: The adjusted mortality rate was calculated, and the LD was calculated using SPSS software Probit analysis. 50 Value and 95% confidence limit.

[0071] The mortality rate of the blank control group at 48 h was 4.5% (≤10%, indicating a valid experiment). Solamelin A's LD50 against southern root-knot nematodes... 50 The value was 8.76 mg / L (95% confidence limit: 6.52-11.34 mg / L). This indicates that the compound Solamelin A provided by this invention exhibits excellent inhibitory activity against nematodes.

[0072] Example 6: Activity test to kill *Strombus erythropterus* Following the standard insecticidal test methods of the Ministry of Agriculture and Rural Affairs of China, the insecticidal activity of compound Solamelin A against Tribolium castaneum was determined using the micro-droplet method. The specific procedures are as follows: (1) Preparation of test solution: Accurately weigh 5 mg of Solamelin A and dissolve it in 0.5 mL of a mixed solvent, which includes acetone and n-hexane, with an acetone:n-hexane ratio of 5:95 (v / v), to prepare a 10 μg / μL stock solution (acetone content 5%). Dilute with n-hexane and set 5 dose gradients, namely 2 μg / μL, 1 μg / μL, 0.5 μg / μL, 0.25, and 0.125 μg / μL, with a final acetone content ≤1% (v / v).

[0073] (2) Test insects: Tribolium castaneum was fed in whole wheat flour. Healthy adults within 2 weeks of emergence were selected, with a body length of 3.5-4.5 mm and a weight of 2-2.5 mg.

[0074] (3) Anesthesia: CO2 anesthesia for 5-8 seconds (the anesthesia time is strictly controlled within 10 seconds to avoid damage).

[0075] (4) Dropping: Using a microsyringe (1 μL specification), accurately drop 0.5 μL of the test drug solution onto the pronotum of the test insect, with a drop volume error of ±0.02 μL. Treat 30 test insects per dose, and repeat 3 times (i.e., 90 insects / dose).

[0076] (5) Blank control: a mixture of hexane and acetone (acetone volume percentage is 1%). Refer to step (4) to drop this solution. The rest of the operation is the same as the test solution.

[0077] (6) Positive control: Accurately weigh 5 mg of 97% rotenone technical grade and prepare rotenone solutions at doses of 5 μg / μL, 2.5 μg / μL, 1.25 μg / μL, 0.625 μg / μL, and 0.3125 μg / μL as in step (1). Drop different doses of rotenone solution as in step (4), with the remaining operations the same as for the test drug solution.

[0078] (7) Culture: After treatment, each group was transferred to a 250 mL wide-mouth bottle, 50 g of whole wheat flour was added, and the bottles were placed in a constant temperature incubator at (27±1)℃, RH 75%±5% and in the dark.

[0079] (8) Investigation: Observe for 72 hours after treatment. If the insect does not react (does not move or attach limbs) when touched with a small brush, it is considered dead. The mortality rate of the blank control is ≤10% to be considered a valid test.

[0080] (9) Statistics: The corrected mortality rate was calculated, and the LD50 value and 95% confidence limit were calculated using SPSS software Probit analysis.

[0081] The mortality rate at 72 h in the blank control group was 6.7% (≤10%, indicating a valid trial). Solamelin A's LD50 against the red flour beetle... 50 The value was 0.87 μg / worm (meaning that when each test worm was precisely administered 0.87 μg of Solamelin A, the mortality rate was 50%, 95% confidence limit: 0.65–1.12 μg / worm), rotenone LD50. 50 The effective value was 3.25 μg / insect (95% confidence limit: 2.48-4.13 μg / insect), with a relative toxicity index of 3.74. This indicates that the compound Solamelin A provided by this invention exhibits significant contact toxicity activity against flour beetles, exceeding the level of the positive control.

[0082] Example 7: Determination of the activity of the maize weevil Following the standard insecticidal test methods of the Ministry of Agriculture and Rural Affairs of China, the insecticidal activity of compound Solamelin A against the maize weevil (Sitophilus oryzae) was determined using the grain dressing method. The specific procedures are as follows: (1) Preparation of test solution: Accurately weigh 50 mg of Solamelin A, dissolve it in 2 mL of acetone to prepare a 25 mg / mL stock solution. Set up 5 concentration gradients, namely 5 mg / kg, 2.5 mg / kg, 1.25 mg / kg, 0.625 mg / kg, and 0.3125 mg / kg (based on wheat mass), with a final acetone content ≤1% (v / v).

[0083] (2) Mixing with medicine: Take 50 g of wheat (moisture content 12-13%, crushed through a 20-mesh sieve), place it in a 250 mL stoppered conical flask, add 1 mL of the test medicine solution, seal and shake vigorously for 5 min to make the test medicine solution evenly adhere to the surface of the grain, and leave it open for 30 min to evaporate the solvent.

[0084] (3) Test insects: Corn weevil (Sitophilus oryzae) was fed with wheat at (28±1)℃ and RH 70%. Healthy adults that had emerged 1-2 weeks prior and weighed 2-2.5 mg were selected.

[0085] (4) Inoculation: Take 50 g of medicated wheat for each treatment and put it into a 500 mL culture bottle. Inoculate 30 adult insects and seal.

[0086] (5) Blank control: a mixture of acetone and n-hexane (acetone volume percentage is 1%). Refer to step (2) to mix the solution with the drug, and the rest of the operation is the same as the test solution.

[0087] (6) Positive control: Weigh 50 mg of 95% malathion technical material accurately, and prepare malathion solutions of 5 mg / kg, 2.5 mg / kg, 1.25 mg / kg, 0.625 mg / kg and 0.3125 mg / kg according to step (1). Mix the malathion solutions of different concentrations with the drug according to step (2), and perform the remaining operations as with the test drug solution.

[0088] (7) Culture: Each group was placed in a constant temperature incubator at (28±1)℃, RH 70%±5% and in darkness, and each treatment was repeated 3 times.

[0089] (8) Investigation: Check the mortality rate 7 days after treatment. Gently touch the insect with a small brush. If there is no reaction, the insect is considered dead. The mortality rate of the blank control is ≤5% to be considered a valid test.

[0090] (9) Statistics: Calculate the adjusted mortality rate and use SPSS software Probit analysis to calculate LD. 50 Value and 95% confidence limit.

[0091] The 7-day mortality rate in the blank control group was 3.5% (≤5%, indicating a valid trial). Solamelin A's LD50 against the maize weevil... 50 The LD50 value was 2.15 mg / kg (95% confidence limit: 1.68–2.73 mg / kg), malathion LD50. 50 The effective value was 3.42 mg / kg (95% confidence limit: 2.71-4.28 mg / kg), with a relative toxicity index of 1.59. This indicates that the compound Solamelin A of this invention exhibits excellent insecticidal activity against maize weevils, exceeding that of the positive control.

[0092] Example 8: Insecticide Activity Test Following the standard insecticidal test methods of the Ministry of Agriculture and Rural Affairs of China, the insecticidal activity of compound Solamelin A against Aedes aegypti was determined using the larval immersion method. The specific procedures are as follows: (1) Preparation of test solution: Accurately weigh 2 mg of Solamelin A, dissolve it in 0.2 mL of acetone to prepare a 10 mg / mL stock solution. Dilute with dechlorinated tap water containing 0.5% (v / v) Tween-80, and set 5 concentration gradients, namely 0.08 mg / L, 0.04 mg / L, 0.02 mg / L, 0.01 mg / L and 0.005 mg / L, with a final acetone content ≤1% (v / v).

[0093] (2) Larval rearing: Aedes aegypti was reared at (26±1)℃, RH 70%, and photoperiod 16 L:8 D. After egg masses were collected and hatched, 7-8 day old fourth instar larvae (body length 6-8 mm, head capsule width 0.8-1 mm) were used for testing.

[0094] (3) Experimental system: Fourth instar larvae were placed in 50 mL beakers (20 larvae per beaker), and the test drug solution was added to 30 mL. Each treatment was repeated 3 times.

[0095] (4) Blank control: Dechlorinated tap water containing acetone and Tween-80 (acetone volume percentage is 1%, Tween-80 volume percentage is 0.5%). Add this solution to the experimental system in step (3), and perform the other operations as in the test solution.

[0096] (5) Incubation: Each group was placed in an artificial climate chamber (26±1℃, RH 70%, 16 L:8 D photocycle) and incubated for 24 h.

[0097] (6) Investigation: After 24 hours, those who are unable to move (sink to the bottom, do not float, and do not respond to mechanical stimulation) are considered dead. The mortality rate of the blank control group is ≤5% to be considered a valid test.

[0098] (7) Statistics: The adjusted mortality rate was calculated, and the LD was calculated using SPSS software Probit analysis. 50 Value and 95% confidence limit.

[0099] The 24-hour mortality rate of the blank control was 4.2% (≤5%, indicating a valid test). Solamelin A's LD50 against Aedes aegypti larvae was also measured. 50 The value was 0.012 mg / L (95% confidence limit: 0.009-0.016 mg / L). This indicates that the compound Solamelin A provided by this invention exhibits insecticidal activity against Aedes mosquito larvae.

[0100] Example 9: Fly-killing activity test determination Following the standard insecticidal test methods of the Ministry of Agriculture and Rural Affairs of China, the insecticidal activity of compound Solamelin A against flies (Musca domestica) was determined using the micro-droplet method. The specific procedures are as follows: (1) Preparation of test solution: Accurately weigh 5 mg of Solamelin A and dissolve it in 0.5 mL of a mixed solvent, which includes acetone and n-hexane, with an acetone:n-hexane ratio of 10:90 (v / v), to prepare a 10 μg / μL stock solution (acetone content 1%). Dilute with n-hexane and set 5 dose gradients, namely 0.6, 0.3, 0.15, 0.075, and 0.0375 μg / μL, with a final acetone content ≤1% (v / v).

[0101] (2) Test insects: Houseflies (Musca domestica) were raised at (26±1)℃, RH 60%, and photoperiod 16 L:8 D, and fed with milk powder and sugar. Healthy female flies that were 3-5 days old and weighed 18-22 mg were selected for testing.

[0102] (3) Anesthesia: Anesthetize with CO2 for 5-8 seconds (until the fly just stops moving), and immediately administer an IV drip.

[0103] (4) Drip administration: Using a micro-drip device (1 μL specification), accurately drip 0.5 μL of the test solution onto the center of the mesothorax of the female fly, with a drip volume error of ±0.02 μL. Each dose was used to treat 30 flies, and the treatment was repeated 3 times (total 90 flies).

[0104] (5) Blank control: a mixture of hexane and acetone (acetone volume percentage is 1%). 0.5 μL of the mixture was dropped according to step (4), and the rest of the operation was the same as the test solution.

[0105] (6) Recovery and feeding: After each group was dripped, it was allowed to recover for 5 minutes (until the solvent evaporated), and then transferred to an insect rearing cage (40 cm × 40 cm × 40 cm). Clean water and cotton balls with 10% sugar water were provided. The cages were then placed in a condition of (26±1)℃, RH 60%, and photoperiod 16 L:8 D.

[0106] (7) Investigation: The mortality rate was counted after 24 hours. The insects were considered dead if their appendages did not move and their bodies did not contract when touched with a small brush. The mortality rate of the blank control group was ≤10% to be considered a valid test.

[0107] (8) Statistics: The adjusted mortality rate was calculated, and the LD was calculated using SPSS software Probit analysis. 50 Value and 95% confidence limit.

[0108] The 24-hour mortality rate of the blank control was 7.3% (≤10%, indicating the experiment was effective). Solamelin A's LD50 against houseflies... 50 The value was 0.16 μg / insect (meaning that when each housefly received 0.16 μg of Solamelin A, the 24-hour mortality rate was 50%, 95% confidence limit: 0.12-0.21 μg / insect). This indicates that the compound Solamelin A provided by this invention exhibits contact toxicity against houseflies.

[0109] In summary, it can be seen that the compound provided by the present invention has the following advantages: (1) Broad insecticidal spectrum and excellent activity: A single spirocyclic sesquiterpene compound can effectively control seven major pests in three major areas: agriculture, storage, and sanitation. Among them, it is effective against peach aphid (LD). 5011.45 mg / L) and Carmine Tetranychus cinnabarinus (LD50) 50 The toxicity of the 16.98 mg / L (LD50) was 2.9 times and 7.2 times that of the positive controls matrine and spirodiclofen, respectively, and it was also effective against the red flour beetle (LD50). 50 0.87 μg / insect) and maize weevil (LD) 50 The activity of 2.15 mg / kg was also significantly better than that of conventional agents such as rotenone and malathion, and its LD50 against southern root-knot nematodes was also significantly higher. 50 The concentration (8.76 mg / L) provides a novel lead structure for plant-derived nematicides, representing a significant breakthrough in achieving "multiple uses from a single agent."

[0110] (2) High environmental and ecological safety: The compound Solamelin A provided by this invention is a natural secondary metabolite derived from eggplant. It is easily degraded, has low residue, no carcinogenic or teratogenic risks, and is friendly to non-target organisms, perfectly matching the green prevention and control and sustainable agricultural development strategy.

[0111] (3) Great value in resistance management: The new spirocyclic skeleton has no cross-resistance with existing pesticides, providing an innovative solution to the increasingly serious problem of pesticide resistance in pests worldwide.

[0112] (4) Excellent economic benefits: The raw materials are widely available, can be extracted or biosynthesized on a large scale, and "one dose can treat multiple insects" significantly reduces the cost of field application, resulting in outstanding economic and social benefits.

[0113] The above experimental results demonstrate that the compounds involved in this invention have good insecticidal activity and a broad insecticidal spectrum, and can be used to prepare plant-derived insecticides. As plant-derived natural products, they have broad application prospects in the field of pesticides.

Claims

1. A spirocyclic sesquiterpene compound, characterized in that, Compounds including those with the structure shown in formula (1), Equation (1).

2. The method for preparing the spirocyclic sesquiterpene compound according to claim 1, characterized in that, Includes the following steps: (1) Using eggplant root as raw material, ethanol extraction was performed to obtain ethanol extract; (2) The ethanol extract was processed by silica gel column chromatography, and gradient elution was performed using hexane-acetone as the eluent. The fraction obtained by elution with a volume ratio of hexane to acetone of 10:1 was collected. (3) The fractions eluted by hexane and acetone collected in step (2) were processed by silica gel column chromatography with petroleum ether-acetone as the eluent and the volume ratio of petroleum ether to acetone being 8:

1. The eluted fractions of the 4th to 5th column volumes were collected. (4) The petroleum ether and acetone eluent fractions collected in step (3) were purified by gel chromatography using a chloroform-methanol system to obtain spirocyclic sesquiterpenoid compounds.

3. The method for preparing the spirocyclic sesquiterpene compound according to claim 2, characterized in that, In step (1), the alcohol extraction is performed 2-4 times, the temperature of each extraction is 25-30℃, and the extraction time is 3-7 days.

4. The method for preparing the spirocyclic sesquiterpene compound according to claim 2 or 3, characterized in that, In step (4), Sephadex LH-20 gel was used for purification.

5. The method for preparing the spirocyclic sesquiterpene compound according to claim 2 or 3, characterized in that, In step (4), the volume ratio of chloroform to methanol in the chloroform-methanol system is 1:0.

8.

6. A pesticide, characterized in that, Includes the spirocyclic sesquiterpene compound of claim 1.

7. The use of the spirocyclic sesquiterpene compound of claim 1 in the preparation of pesticides.

8. The use of the spirocyclic sesquiterpene compound of claim 1 or the pesticide of claim 6 in the control of pests.

9. The application according to claim 8, characterized in that, The pests mentioned are agricultural pests, storage pests, or sanitary pests.

10. The application according to claim 9, characterized in that, The pests are selected from one or more of the following: aphids, mites, root-knot nematodes, red flour beetles, corn weevils, mosquitoes, and flies.

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