A novel plant-derived ecdysone agonist from sida cordifolia and its preparation method and application

Abstract

This invention discloses a novel plant-derived ecdysone antagonist derived from Dryopteris crassirhizoma, its preparation method, and its application, belonging to the field of biological control of agricultural pests and green pesticide technology. The novel plant-derived ecdysone antagonist disclosed in this invention has the molecular formula C0.05. 21 H 24 O8. This invention discloses a novel plant-derived ecdysone antagonist derived from Dryopteris crassirhizoma, targeting the insect's unique developmental system. It exhibits high safety for non-target organisms such as mammals and pollinating insects, and is readily degradable in the environment, perfectly meeting the dual requirements of agricultural product safety and ecological protection. This invention not only provides a novel pesticide but also establishes a complete technical system from target discovery and efficient screening to green application.

Description

Technical Field

[0001] This invention relates to the field of biological control of agricultural pests and green pesticides, and more specifically to a novel plant-derived ecdysone antagonist derived from Dryopteris crassirhizoma, its preparation method, and its application. Background Technology

[0002] Insects are the most diverse group of animals on Earth, and many species are important agricultural pests, posing a serious threat to global food security. For a long time, chemical pesticides have been the primary means of pest control, playing an irreplaceable role in ensuring crop yields. However, traditional neurotoxic pesticides, represented by organophosphates, pyrethroids, and neonicotinoids, have revealed many serious problems during long-term large-scale use due to their broad-spectrum toxicity and non-specific mechanisms of action. First and foremost, the most prominent problem is the emergence and development of pesticide resistance in pests. Since the first report of insect pesticide resistance in the early 20th century, more than 500 insect and mite species have developed resistance to one or more commonly used pesticides. Taking the brown planthopper, a major rice pest in my country, as an example, its resistance to mainstream pesticides such as imidacloprid and thiamethoxam has reached hundreds of times in some areas, leading to a significant decrease in control effectiveness and creating a vicious cycle of "increased pesticide use - intensified resistance." Secondly, traditional pesticides have significant direct or sublethal effects on non-target organisms, especially pollinating insects (such as bees), natural enemies (such as ladybugs and parasitic wasps), and aquatic organisms, severely damaging the biodiversity and natural regulation capacity of farmland ecosystems. Furthermore, pesticide residues accumulate through the food chain, posing a potential threat to food safety and human health. Simultaneously, pesticide components not utilized by their targets enter the environment, causing persistent environmental problems such as soil compaction and water pollution.

[0003] Faced with these challenges, the international community has put forward new requirements for pesticide research and application: "green, low-toxicity, and highly selective." Against this backdrop, third-generation insecticides—insect growth regulators (IGRs)—that act on the unique physiological systems of insects have emerged. IGRs do not directly kill adult insects, but rather achieve effective and sustainable control of their populations by interfering with or inhibiting key physiological processes such as growth, development, molting, metamorphosis, and reproduction. Due to their unique mechanism of action, low toxicity to mammals, and good environmental compatibility, IGRs are considered an important alternative to traditional insecticides and a core component of modern integrated pest management strategies.

[0004] Insect growth and metamorphosis is a highly programmed and complex biological process, primarily regulated by the synergistic action of two endogenous hormones: juvenile hormone (JH) and ecdysone (20-Hydroxyecdysone, 20E). 20E is the core steroid hormone initiating and driving insect molting and metamorphosis. During larval development, the titer of 20E in the hemolymph exhibits periodic peaks, each peak precisely initiating a molting process, allowing the larva to grow and enter the next instar. In the final larval instar, a unique 20E peak triggers the metamorphosis from larva to pupa (holometabolous insects) or adult (hemimetabolous insects). This series of complex morphological remodeling processes, including the dissociation of larval-specific tissues, the development of adult organs, and the synthesis of new epidermis, are all completed under the precise temporal regulation of 20E.

[0005] At the molecular level, the regulatory mechanism of 20E has been thoroughly elucidated. After entering the cell, 20E binds to its specific receptor in the cell nucleus—the ecdysone receptor (EcR)—and its heterodimeric chaperone—the ultraspiracle (USP). It then specifically recognizes and binds to the ecdysone response element (EcRE) in the promoter region of downstream target genes, thereby initiating a series of early response genes (such as…). Broad-Complex , E74 , E75 Transcription of hormones (such as hormone binding and gene activation) is crucial for the development of novel selective insecticides. These early transcription factors further regulate the expression of numerous late-stage effector genes, ultimately forming a sophisticated gene expression network that performs specific physiological functions such as molting, metamorphosis, autophagy, and apoptosis. This complete signaling pathway, from hormone binding to gene activation, provides an ideal molecular target for the development of novel selective insecticides.

[0006] Based on the understanding of the 20E signaling pathway, the most successful commercial examples are diazinon insecticides, such as tebufenozide, methoxyfenozide, cyclotebufenozide, chlorfenapyr, and furazolidone. These compounds are 20E agonists; they can bind to the EcR / USP receptor complex and abnormally activate molting signals, causing larvae to prematurely and inappropriately enter the molting process while juvenile hormone levels are still high, ultimately leading to death due to incomplete molting. However, after widespread use, the inherent limitations of diazinon insecticides have gradually become apparent. The primary problem is the homogenization of their chemical structures; all commercial varieties share a highly similar tert-butyldibenzoylhydrazine core, resulting in a single mode of action. Field monitoring data has confirmed that several important lepidopteran pests, such as the beet armyworm and diamondback moth, have developed significant levels of resistance to tebufenozide and similar insecticides, and have also shown reduced sensitivity to methoxyfenozide. This phenomenon of "structural homogenization" means that once pests develop resistance to one pesticide, they may also develop cross-resistance to all pesticides of the same type, significantly shortening the market life cycle of such pesticides. Therefore, the escalating risk of pest resistance urgently requires us to move beyond the existing agonist research and development approach and find novel compounds with distinct mechanisms of action.

[0007] In contrast to the mechanism of action of agonists, ecdysone antagonists, as a type of IGR that has not yet been fully explored and utilized, show great potential for development. Antagonists can bind to EcR / USP receptors, but do not trigger the activation of downstream signaling pathways. Instead, they competitively occupy the hormone-binding site, blocking the normal binding of natural 20E to its receptor, thus "freezing" the entire ecdysone signaling pathway. This effect prevents insects from initiating the molting process at critical developmental stages, leading to developmental arrest, molting disorders, deformities, and even death.

[0008] Because antagonists and agonists have fundamentally different modes of action, they may still be effective against pest populations that have developed resistance to dihydrazides, providing a novel approach to addressing existing resistance problems. Furthermore, screening antagonists from natural products, particularly plant secondary metabolites with rich chemical diversity, is an important strategy for discovering lead compounds with novel structural skeletons, which can further reduce the risk of cross-resistance with existing agents.

[0009] Therefore, providing a novel plant-derived ecdysone antagonist derived from Dryopteris crassirhizoma, along with its preparation method and application, is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0010] In view of this, the present invention provides a novel plant-derived ecdysone antagonist derived from Dryopteris crassirhizoma, its preparation method and application.

[0011] This invention targets insect molting hormone receptors as specific targets and aims to develop plant-derived antagonists that interfere with the normal development of insects. Essentially, it is a rational pesticide design based on target action.

[0012] To achieve the above objectives, the present invention adopts the following technical solution:

[0013] A novel plant-derived ecdysone antagonist derived from Dryopteris crassirhizoma has the following chemical structural formula: .

[0014] Furthermore, the preparation method of the ecdysone antagonist includes the following steps: (1) Ethanol extraction: The rhizome of Dryopteris crassirhizoma was crushed and sieved, the powder was weighed, 8 times the volume of 95% ethanol was added, and the mixture was refluxed at 55°C for 2 h and filtered. The residue was extracted once more, the filtrates were combined, and the extract was concentrated under reduced pressure at 50°C to obtain a paste. The crude extract was then freeze-dried. (2) Liquid-liquid extraction: The crude extract was dispersed in distilled water and extracted three times each with equal volumes of petroleum ether, ethyl acetate, and n-butanol. The extracts were combined, concentrated and dried under reduced pressure to obtain petroleum ether phase, ethyl acetate phase, n-butanol phase and aqueous phase. The ecdysone antagonistic activity was tested to determine that the active fraction was the ethyl acetate phase. (3) Silica gel column chromatography: The ethyl acetate phase was eluted by a 200-300 mesh normal phase silica gel column with a dichloromethane-methanol gradient of 100:0 → 0:100 v / v. One fraction was collected every 500 mL, and the activity of each fraction was detected. The active fraction was divided into 10 components. (4) Gel column chromatography: Component 10 was further subjected to Sephadex LH-20 gel column elution with dichloromethane-methanol 100:0→0:100, v / v, and each component was collected and its activity was detected. The activity was enriched in components 10-7 to 10-12. (5) After combining the 10⁻⁷ to 10⁻¹² components, the mixture was purified by semi-preparative HPLC and the target peak was collected. The ecdysone antagonist with the strongest antagonistic activity was obtained by detection.

[0015] Furthermore, the application of the ecdysone antagonist in the biological control of agricultural pests.

[0016] Furthermore, the application of the molting hormone antagonist in the preparation of insect growth regulators.

[0017] As can be seen from the above technical solution, compared with the prior art, this invention discloses a novel plant-derived ecdysone antagonist derived from Dryopteris crassirhizoma, its preparation method, and its application. The ecdysone antagonist of this invention aims to systematically solve multiple technical bottlenecks in the current field of pest control: First, it provides a fundamental solution to the increased pesticide resistance and cross-resistance phenomenon in pests caused by the structural homogenization of existing ecdysone agonists (such as dihydrazides); second, it breaks through the innovation dilemma of single mechanism of action, providing a lead compound for a new paradigm of action in pesticide research and development; finally, it responds to the needs of green agriculture and promotes the replacement of highly toxic chemical pesticides. Compared to existing technologies, the core advantages of this invention are reflected in three dimensions: Firstly, in terms of its mechanism of action, it uniquely blocks rather than mimics the ecdysone signaling pathway through "competitive antagonism," thus disrupting the pest development process at its source. It maintains high efficacy even in populations that have developed agonist resistance, making it a strategic tool for resistance management. Secondly, at the research and development stage, it discovers novel chemical skeletons from plant-derived natural products, breaking through the limitations of existing parent nuclei and laying the material foundation for creating pesticides with independent intellectual property rights, achieving a paradigm shift from "simulation" to "intervention." Simultaneously, this antagonist targets the insect's unique developmental system, exhibiting high safety for non-target organisms such as mammals and pollinators, and is easily degraded in the environment, perfectly meeting the dual needs of agricultural product safety and ecological protection. This innovation not only provides a novel pesticide but also constructs a complete technological system from target discovery and efficient screening to green application.

[0018] The plant-derived ecdysone antagonist obtained in this invention (1) can be used to develop novel, efficient, and low-toxicity insect growth regulators. It interferes with or blocks the normal molting and metamorphosis of pests by antagonizing the binding of ecdysone to its receptor, thereby achieving chemical control of pests. Compared with existing dihydrazide agonists, the compound of this invention has a novel plant-derived skeleton, which is expected to overcome the problem of cross-resistance caused by target mutations and provide a new mechanism of action for resistance management. It can be used directly as the active ingredient of plant-derived pesticides or as a parent structure for further structural modification and optimization to develop a series of environmentally friendly pesticide varieties. (2) It can be used as a tool molecule to study the ecdysone signal transduction pathway, the molecular regulatory mechanism of insect metamorphosis, and the structure-activity relationship of the interaction between the ecdysone receptor (EcR) and its ligand. By utilizing its antagonistic properties, ecdysone signaling can be "turned off" or inhibited at the cellular or individual level, thereby verifying the function of related genes in reverse. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0020] Figure 1 Flowchart for screening ecdysone antagonists.

[0021] Figure 2 This study aimed to induce the EcRE reporter gene response to 20E in Kc cells.

[0022] Figure 3 The inhibitory effect of each extract of Dryopteris crassirhizoma on 20E-induced luciferase activity was investigated. The extracts were: Ethanolex tract (crude ethanol extract); petroleum ether (petroleum ether phase); ethyl acetate (ethyl acetate phase); n-butanol (n-butanol phase); and water (aqueous phase).

[0023] Figure 4 The effect of silica gel column chromatography fraction of Dryopteris crassirhizoma extract on 20E-induced luciferase activity was investigated.

[0024] Figure 5 The inhibitory effect of the gel column chromatography fraction of Dryopteris crassirhizoma extract on 20E-induced luciferase activity was investigated.

[0025] Figure 6 This is a semi-preparative HPLC purified compound.

[0026] Figure 7 To detect the activity of isolated monomeric compounds.

[0027] Figure 8 Here is the chemical structural formula of the active compound A3.

[0028] Figure 9 This describes the dose-dependent inhibition of 20E-induced luciferase activity by the active monomeric compound.

[0029] Figure 10 The diagram shows the developmental delay and abnormal phenotypes in Drosophila caused by the active monomer compound A3.

[0030] Figure 11 The active monomeric compound A3 caused developmental deformities and death in the Asian corn borer. A: Phenotypic diagram of developmental deformities caused by active monomeric compound A3 in the Asian corn borer; B: Diagram of adult emergence obstruction, inability to shed pupal shells normally, and wing deformities; C: Percentage at each stage. Detailed Implementation

[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] The key points of the plant-derived ecdysone antagonist screening technology involved in this invention are as follows: Figure 1 As shown. The screening strategy employed activity-guided separation, using Drosophila Kc cells transfected with an EcRE-driven luciferase reporter gene as the screening model. Through activity screening of a large number of plant crude ethanol extracts, it was found that the ethanol extract of Dryopteris crassirhizoma tuber exhibited significant ecdysone antagonistic activity, effectively inhibiting 20E-induced luciferase expression.

[0033] To identify the active ingredient, the crude extract of Dryopteris crassirhizoma tuber was sequentially purified by silica gel column chromatography, gel column chromatography (Sephadex LH-20), and semi-preparative high-performance liquid chromatography (HPLC). During the separation process, the activity of each fraction was monitored and confirmed in real time using the aforementioned cell reporter system, ultimately yielding a high-purity active compound, named A3.

[0034] The structure of compound A3 was identified using spectroscopic methods: 1H NMR and 1C NMR. 13 CNMR revealed that its characteristic signal exhibited high symmetry, and the chemical shift values ​​conformed to the structural features of multi-substituted benzene rings and carbonyl groups. Combined with the precise molecular weight information provided by high-resolution mass spectrometry (HRMS), compound A3 was ultimately identified as a phloroglucinol derivative with a symmetrical dimer structure. The skeleton of this compound is significantly different from existing dihydrazide-based synthetic insecticides, representing a novel class of naturally derived ecdysone antagonists.

[0035] See patent 202311591973.2 for the pGL3-EcRE-Fluc reporter plasmid.

[0036] Example 1 1) Establishment of an EcRE reporter detection system in Drosophila Kc cells Drosophila Kc cells in the logarithmic growth phase were seeded into 24-well plates at a density of 2 × 10⁶ cells / well. 5Cells / wells were cultured overnight at 28°C to ensure full adhesion. Using TransIntro EL transfection reagent (Beijing TransGen Biotech Co., Ltd.), pGL3-EcRE-Fluc reporter plasmid (400 ng / well) and pRL-SV40 internal control plasmid (40 ng / well, Promega, USA) were co-transfected into Kc cells. pRL-SV40 constitutively expresses René luciferase and was used to correct for transfection efficiency and cytotoxicity. 24 h after transfection, the culture medium was aspirated, and each well was incubated with 1 μmol / L 20E and 100 ppm of the crude extract or isolated fraction, and incubated at 28°C for another 4 h. Subsequently, the culture medium was aspirated, and luciferase activity was measured using a dual-luciferase reporter gene assay kit (Shanghai Yisheng Biotechnology Co., Ltd.). Add 200 μL of lysis buffer to each well, incubate at room temperature with shaking for 30 min, and take 10 μL of the lysis buffer. Measure the luminescence values ​​of firefly luciferase (Fluc) and kidney luciferase (Rluc) sequentially using a microplate reader (Tecan Infinite 200 PRO, Switzerland). Calculate the Fluc / Rluc ratio and normalize it with the solvent control group as 1 to calculate the relative luciferase activity. For antagonistic activity evaluation, different test samples were added to a medium containing 1 μmol / L 20E. A significant decrease in the Fluc / Rluc ratio indicated that the sample had ecdysone antagonistic activity. The inhibition rate was calculated using the following formula: Inhibition rate (%) = (1 - Fluc / Rluc value of the treatment group / Fluc / Rluc value of the 20E group) × 100%. Figure 2 To validate the activity of the EcRE reporter gene detection system, the results showed that, compared with the DMSO control group, the fluorescence ratio of the 1 μmol / L 20E treatment group was significantly increased to 30.5, and the induction fold was approximately 30-fold, indicating that the reporter system was successfully established and can be used for subsequent screening of ecdysone antagonists.

[0037] 2) Preparation and activity tracking separation of crude plant extracts Plant material: Rhizome of Dryopteris crassirhizoma Nakai., crushed and passed through a 40-mesh sieve.

[0038] Ethanol extraction: Weigh 100 g of powder, add 8 times the volume of 95% ethanol, reflux at 55℃ for 2 h, and filter; repeat the extraction once with the residue, combine the filtrates, concentrate under reduced pressure at 50℃ to extract, and freeze dry to obtain crude extract.

[0039] Liquid-liquid extraction: The crude extract was dispersed in an appropriate amount of distilled water and extracted three times each with equal volumes of petroleum ether, ethyl acetate, and n-butanol. The extracts were combined, concentrated under reduced pressure, and dried to obtain the petroleum ether phase, ethyl acetate phase, n-butanol phase, and aqueous phase. The 20E antagonistic activity of each phase was detected using the above reporter gene system to determine the active site (e.g., the ethyl acetate phase).

[0040] Silica gel column chromatography: The active fraction (ethyl acetate phase) was eluted by a normal-phase silica gel column (200-300 mesh) with a gradient of dichloromethane-methanol (100:0→0:100, v / v). One fraction was collected every 500 mL, and the activity of each fraction was determined.

[0041] Gel column chromatography: The active fraction was further eluted with dichloromethane-methanol (100:0→0:100, v / v) on a Sephadex LH-20 gel column, and the fractions were collected and their activity was determined.

[0042] Figure 3 The antagonistic activity of each extract phase (100 ppm) of the crude ethanol extract of Dryopteris crassirhizoma was detected in the presence of 1 μmol / L 20E after liquid-liquid partition extraction. The results showed that the ethyl acetate phase had the strongest antagonistic activity.

[0043] After separation of the ethyl acetate phase by silica gel column chromatography, the antagonistic activity of each fraction (100 ppm) was detected. The activity was mainly concentrated in fraction 10, which had the highest inhibition rate. Figure 4 ).

[0044] After further separation of component 10 by Sephadex LH-20 gel column chromatography, the antagonistic activity of each sub-component (100 ppm) was detected. The activity was enriched in components 10⁻⁷ to 10⁻¹². Figure 5 ).

[0045] 3) Isolation of active monomers and verification of antagonistic activity The 10⁻⁷ to 10⁻¹² fractions were combined and purified by semi-preparative HPLC (Agilent ZORBAX SB-C18 column, 9.4 × 250 mm, 5 μm). The mobile phase was acetonitrile-water (1:1, containing 0.1% trifluoroacetic acid), the flow rate was 3 mL / min, and the detection wavelength was 254 nm. Three main peaks were observed, and the target peaks were manually collected and labeled A1, A2, and A3. Figure 6 ). Reporter gene assays were performed on the three compounds, and compound A3 was found to have the strongest antagonistic activity ( ). Figure 7 ).

[0046] The structure of compound A3 was identified using nuclear magnetic resonance (NMR) spectroscopy, including one-dimensional NMR (NMR). 1 H NMR, 13Correlation analysis of C10 NMR and two-dimensional NMR (HSQC, HMBC) data, combined with high-resolution mass spectrometry (HRMS) data, finally determined its chemical structure. Specific spectral data and interpretations are shown in Table 1.

[0047] Table 1. Nuclear magnetic resonance spectral data of compound A3 (600 MHz, 150 MHz, DMSO-d6)

[0048] Based on the above spectroscopic data analysis, compound A3 was identified as a highly symmetrical dimeric phloroglucinol derivative, consisting of two phloroglucinol units linked at the C-1 position via a methylene bridge (C-7). Each unit has a propionyl side chain attached to the C-3 position, with hydroxyl substitutions at C-2, C-4, and C-6, and an aromatic hydrogen retained at the C-5 position. Comparison with literature data confirmed its identity as Methylene-bis-phlorobutyrophenone, with the molecular formula C... 21 H 24 O8, its chemical structural formula is as follows Figure 8 As shown in the figure. This structural framework is significantly different from existing dihydrazide-based synthetic insecticides, representing a novel class of naturally derived ecdysone antagonists.

[0049] To verify the ecdysone antagonistic activity of compound A3, different concentrations (0.01, 0.1, 1, 10, and 100 μmol / L) of compound A3 were added to a culture medium containing 1 μmol / L 20E, and its inhibitory effect on 20E-induced luciferase activity was detected. The results showed that the compound inhibited 20E-induced luciferase activity in a dose-dependent manner, with an IC50 value of [missing value]. 50 The value was 0.3069 ± 0.0712 μmol / L ( Figure 9 ).

[0050] Example 2 The ecdysone antagonist obtained by this invention was tested for its insecticidal activity in Drosophila melanoptera and Asian corn borer, respectively.

[0051] Feed-to-toxicity method (Drosophila): Compound A3 was dissolved in 95% ethanol and mixed into artificial feed at final concentrations of 0.25, 0.5, and 1 mg / mL. Third-instar Drosophila larvae were fed this mixture. Each treatment had three replicates, with 20 larvae per replicate. Feed containing an equal volume of ethanol served as a control. Larval pupation was recorded, and phenotypes were observed.

[0052] The results showed that 48 hours after treatment, the control group pupated normally, while the treatment group experienced developmental arrest, remaining as 3rd instar larvae, and ultimately none of them pupated. Figure 10 Compound A3 can inhibit fruit fly molting and pupation.

[0053] Abdominal drip method (Asian corn borer): Fifth-instar Asian corn borer larvae were selected, and 2 μL of compound A3 solution (dissolved in acetone, concentrations of 0.125, 0.25, and 0.5 μg / μL) at different concentrations were dripped onto the abdominal dorsal plate using a pipette. The final treatment volume was 0.25, 0.5, and 1 μg, with an equal volume of acetone as a control. Each treatment consisted of 15 larvae, repeated three times. After treatment, pupation, phenotypic changes, and emergence were observed, and pupation time was recorded.

[0054] The results showed that some treatment groups exhibited deformed phenotypes in their larvae, including melanization, incomplete molting, and larval-pupa intermediates. Figure 11 A), and adult emergence is hindered, they cannot shed their pupal shells normally, and the rate of wing deformities is high ( Figure 11 B), final insecticidal rate >80% ( Figure 11 C). All of the above phenotypes show a certain degree of concentration dependence.

[0055] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A novel plant-derived ecdysone antagonist derived from Dryopteris crassirhizoma, characterized in that, Its chemical structural formula is as follows: 。 2. The method for preparing the ecdysone antagonist according to claim 1, characterized in that, Includes the following steps: (1) Ethanol extraction: The rhizome of Dryopteris crassirhizoma was crushed and sieved, the powder was weighed, 8 times the volume of 95% ethanol was added, and the mixture was refluxed at 55°C for 2 h and filtered. The residue was extracted once more, the filtrates were combined, and the extract was concentrated under reduced pressure at 50°C to obtain a paste. The crude extract was then freeze-dried. (2) Liquid-liquid extraction: The crude extract was dispersed in distilled water and extracted three times each with equal volumes of petroleum ether, ethyl acetate and n-butanol. The extracts were combined and concentrated under reduced pressure and dried to obtain petroleum ether phase, ethyl acetate phase, n-butanol phase and aqueous phase. The antagonistic activity of ecdysone was detected, and the active fraction was determined to be the ethyl acetate phase. (3) Silica gel column chromatography: The ethyl acetate phase was eluted by a 200-300 mesh normal phase silica gel column with a dichloromethane-methanol gradient of 100:0 → 0:100 v / v. One fraction was collected every 500 mL, and the activity of each fraction was detected. The active fraction was divided into 10 components. (4) Gel column chromatography: Component 10 was further subjected to Sephadex LH-20 gel column elution with dichloromethane-methanol 100:0→0:100, v / v, and each component was collected and its activity was detected. The activity was enriched in components 10-7 to 10-12. (5) After combining the 10⁻⁷ to 10⁻¹² components, the mixture was purified by semi-preparative HPLC and the target peak was collected. The ecdysone antagonist with the strongest antagonistic activity was obtained by detection.

3. The application of the ecdysone antagonist as described in claim 1 in the biological control of agricultural pests.

4. The application of the ecdysone antagonist according to claim 1 in the preparation of insect growth regulators.

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