Four insecticidal active compounds in chrysanthemum, and preparation method and application thereof

Abstract

The application belongs to the technical field of new chrysanthemum compounds, and provides four kinds of insecticidal active compounds in chrysanthemum flowers, and a preparation method and application thereof.The insecticidal active compounds provided by the application have the following structures.Tanacetum A and Tanacetum B belong to lignan compounds, Tanacetum C belongs to monoterpenoid ester compounds, and Tanacetum D belongs to sesquiterpene compounds.Tanacetum A-D enriches the types of natural chrysanthemic acid, and shows significant insecticidal activity on aphids, especially on corn aphids, which is of great significance for developing new biological insecticides.The application further provides a preparation method of the insecticidal active compounds described in the technical solution, and the preparation method provided by the application is simple to operate.The application uses chrysanthemum flowers as raw materials for extraction, and improves the resource utilization of chrysanthemum flowers.

Description

Technical Field

[0001] This invention relates to the field of novel pyrethrum compounds, and more particularly to insecticidal active compounds, their preparation methods, and applications. Background Technology

[0002] Pyrethrum is a perennial herbaceous plant belonging to the genus Artemisia in the family Asteraceae. It is a natural source of pyrethrin and has long been known as a natural insecticide. Its active ingredient consists of six components called pyrethrin: pyrethrin I, pyrethrin II, cinerin I, cinerin II, jasmoline I, and jasmoline II. Natural pyrethrin is considered an ideal insecticide due to its green, efficient, safe, environmentally friendly, and residue-free properties.

[0003] Over the years, to overcome the shortcomings of natural pyrethroids, such as poor photostability and high synthesis difficulty, a large number of chemically synthesized pyrethroids have been developed through stereoconfiguration analysis and structural modification of natural pyrethroids. Compared with natural pyrethroids, the photostability of chemically synthesized pyrethroids has been greatly improved. However, pests easily develop resistance to them, and they are highly toxic to mammals, fish, and aquatic animals.

[0004] With the deepening of agricultural mechanization and large-scale production, the demand for highly efficient biological pesticides continues to grow. While pyrethroid compounds, currently the mainstream insecticides, possess broad-spectrum insecticidal activity, their complex synthesis processes lead to numerous problems such as resistance, high residues, and ecotoxicity. Therefore, the development of pesticides derived from natural plants has become a key focus. Pyrethrum, as a traditional medicinal plant, possesses the active ingredient pyrethrin, which exhibits rapid knockdown, good biodegradability, and safety for non-target organisms. In recent years, through modern separation techniques and activity screening, several novel insecticidal active ingredients have been discovered in pyrethrum. These compounds retain the safety advantages of natural products, demonstrating superior insecticidal activity and environmental compatibility. This provides an innovative approach to solving the problems of chemical pesticide residues and resistance, and also provides important theoretical basis for the development of novel plant-derived insecticides, promoting the sustainable development of green pest control and agricultural product quality and safety. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide insecticidal active compounds, their preparation methods, and applications. The insecticidal active compounds provided by this invention enrich the variety of natural pyrethroids and exhibit excellent killing effects against aphids.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides an insecticidal compound having the following structure:

[0008]

[0009] This invention also provides a method for preparing the insecticidal active compound described in the above technical solution, comprising the following steps:

[0010] Pyrethrum flowers were soaked in an ethanol solution for extraction to obtain a crude extract.

[0011] The crude extract was extracted with ethyl acetate to obtain crude extracts of Tanacetum A-D.

[0012] The crude extracts of Tanacetum A-D were eluted by silica gel column chromatography, and the eluents were collected and named Fr1, Fr2, Fr3, Fr4 and Fr5 in sequence. The eluent for the silica gel column chromatography was a chloroform-methanol system, in which the volume ratio of chloroform to methanol was 100:0 to 5:1.

[0013] The Fr2 was subjected to a first silica gel column chromatography to obtain an eluent containing Tanacetum A-B;

[0014] The eluent containing Tanacetum A-B was purified by first dextran gel column chromatography to obtain Tanacetum A and the mixture of Tanacetum A-B, respectively.

[0015] The Tanacetum A-B mixture was eluted by high performance liquid chromatography to obtain Tanacetum B;

[0016] The Fr3 was subjected to a second silica gel column chromatography to obtain an eluent containing Tanacetum C;

[0017] The eluent containing Tanacetum C was purified by second dextran gel column chromatography to obtain Tanacetum C;

[0018] The Fr4 was subjected to a third silica gel column chromatography to obtain an eluent containing Tanacetum D;

[0019] The eluent containing Tanacetum D was purified by third-glucan gel column chromatography to obtain Tanacetum D.

[0020] Preferably, the pyrethrum is the pyrethrum after essential oil extraction;

[0021] The volume concentration of the ethanol solution is 70-90%, and the mass-to-volume ratio of the pyrethrum to the ethanol solution is (15-16) kg: (50-55) L; the soaking time is 12-36 h.

[0022] The extraction is performed 2 to 4 times, with each extraction at a temperature of 30 to 80°C and a time of 10 to 40 minutes.

[0023] After extraction, the process further includes solid-liquid separation of the obtained extraction system to obtain an extract; and concentration of the extract under reduced pressure to obtain the crude extract.

[0024] Preferably, the mass-to-volume ratio of the crude extract to ethyl acetate is (780–830) g: (5–5.5) L.

[0025] Preferably, during the silica gel column chromatography elution process, the volume ratio of chloroform to methanol in the chloroform-methanol system is 100:0, 100:1, 50:1, 80:1, 10:1, and 5:1, respectively.

[0026] Preferably, the eluent for the first silica gel column chromatography is a first petroleum ether-ethyl acetate system, wherein the volume ratio of petroleum ether to ethyl acetate in the first petroleum ether-ethyl acetate system is 80:1.

[0027] Preferably, the eluent for the first dextran gel column chromatography purification is methanol;

[0028] The eluent used in the high-performance liquid chromatography is an acetonitrile-water system, wherein the volume ratio of acetonitrile to water in the acetonitrile-water system is 75:25.

[0029] Preferably, the eluent for the second silica gel column chromatography is a second petroleum ether-ethyl acetate system, wherein the volume ratio of petroleum ether to ethyl acetate in the second petroleum ether-ethyl acetate system is 50:1.

[0030] The eluent for the second dextran gel column chromatography purification was methanol.

[0031] Preferably, the eluent for the third silica gel column chromatography is a third petroleum ether-ethyl acetate system, wherein the volume ratio of petroleum ether to ethyl acetate in the third petroleum ether-ethyl acetate system is 50:1.

[0032] The eluent used for the third dextran gel column chromatography purification was methanol.

[0033] The present invention also provides the application of the insecticidal active compounds described in the above technical solution in killing aphids.

[0034] This invention provides an insecticidal compound.

[0035] In this invention, Tanacetum A-D are secondary metabolites of pyrethrum. Tanacetum A and Tanacetum B belong to the lignan class of compounds, enriching the diversity of lignan compounds; Tanacetum C belongs to the monoterpene ester class, enriching the diversity of monoterpene ester compounds; and Tanacetum D belongs to the sesquiterpene class of compounds. Tanacetum A-D enriches the types of natural pyrethrins and exhibits significant insecticidal activity against aphids, especially corn aphids, which is of great significance for the development of novel biological insecticides. Experiments show that, using the standard insecticidal test method of the Ministry of Agriculture and Rural Affairs, compounds Tanacetum A-D exhibit significant insecticidal activity against aphids, with lethal median concentrations (LC50 values) of 6.856 mg / L, 7.932 mg / L, 33.160 mg / L, and 9.311 mg / L, respectively, while the lethal median concentration (LC50 value) of the positive control imidacloprid is 25.856 mg / L.

[0036] This invention also provides a method for preparing the insecticidal active compounds described in the above-mentioned technical solution. Tanacetum A-D of this invention are secondary metabolites of pyrethrum, obtained through ethanol solution soaking and ethyl acetate extraction. The preparation method provided by this invention has a short cycle, mild conditions, few byproducts, strong stereoselectivity, low cost, and is easy to industrialize. This invention uses pyrethrum after essential oil extraction as raw material, improving the resource utilization of pyrethrum, which not only meets environmental protection and low-carbon requirements but also enables the development of novel and highly efficient biological pesticides. The preparation method provided by this invention is simple and easy to implement, easily industrialized, and the compounds are stable and fully utilize pyrethrum resources. Attached Figure Description

[0037] Figure 1 The HR-ESI-MS spectrum of the compound Tanacetum A of this invention is shown below.

[0038] Figure 2 The compound Tanacetum A of this invention 1 H-NMR spectrum;

[0039] Figure 3 The compound Tanacetum A of this invention 13 C-NMR;

[0040] Figure 4 The HMBC spectrum of the compound Tanacetum A of this invention is shown below.

[0041] Figure 5 The compound Tanacetum A of this invention 1 H- 1 H COSY spectrum.

[0042] Figure 6 The HR-ESI-MS spectrum of the compound Tanacetum B of this invention is shown below.

[0043] Figure 7 The compound Tanacetum B of this invention 1 H-NMR spectrum;

[0044] Figure 8 The compound Tanacetum B of this invention 13 C-NMR;

[0045] Figure 9 The HMBC spectrum of the compound Tanacetum B of this invention is shown below.

[0046] Figure 10 The compound Tanacetum B of this invention 1 H- 1 H COSY spectrum;

[0047] Figure 11 The HR-ESI-MS spectrum of the compound Tanacetum C of this invention is shown below.

[0048] Figure 12 The compound Tanacetum C of this invention 1 H-NMR spectrum;

[0049] Figure 13 The compound Tanacetum C of this invention 13 C-NMR;

[0050] Figure 14 The HMBC spectrum of the compound Tanacetum C of this invention is shown below.

[0051] Figure 15 The compound Tanacetum C of this invention 1 H- 1 H COSY spectrum;

[0052] Figure 16 The HR-ESI-MS spectrum of the compound Tanacetum D of this invention is shown below.

[0053] Figure 17 The compound Tanacetum D of this invention 1 H-NMR spectrum;

[0054] Figure 18 The compound Tanacetum D of this invention 13 C-NMR

[0055] Figure 19 The HMBC spectrum of the compound Tanacetum D of this invention is shown below.

[0056] Figure 20 The compound Tanacetum D of this invention 1 H- 1 H COSY spectrum; Detailed Implementation

[0057] This invention provides an insecticidal compound having the following structure:

[0058]

[0059] In this invention, Tanacetum A and Tanacetum B belong to the lignan class of compounds, Tanacetum C belongs to the monoterpene ester class of compounds, and Tanacetum D belongs to the sesquiterpene class of compounds. Tanacetum A-D enriches the variety of natural pyrethrins and exhibits significant insecticidal activity against aphids, especially corn aphids, which is of great significance for the development of novel biological insecticides.

[0060] This invention also provides a method for preparing the insecticidal active compound described in the above technical solution, comprising the following steps:

[0061] Pyrethrum flowers were soaked in an ethanol solution for extraction to obtain a crude extract.

[0062] The crude extract was extracted with ethyl acetate to obtain crude extracts of Tanacetum A-D.

[0063] The crude extracts of Tanacetum A-D were eluted by silica gel column chromatography, and the eluents were collected and named Fr1, Fr2, Fr3, Fr4 and Fr5 in sequence. The eluent for the silica gel column chromatography was a chloroform-methanol system, in which the volume ratio of chloroform to methanol was 100:0 to 5:1.

[0064] The Fr2 was subjected to a first silica gel column chromatography to obtain an eluent containing Tanacetum A-B;

[0065] The eluent containing Tanacetum A-B was purified by first dextran gel column chromatography to obtain Tanacetum A and the mixture of Tanacetum A-B, respectively.

[0066] The Tanacetum A-B mixture was eluted by high performance liquid chromatography to obtain Tanacetum B;

[0067] The Fr3 was subjected to a second silica gel column chromatography to obtain an eluent containing Tanacetum C;

[0068] The eluent containing Tanacetum C was purified by second dextran gel column chromatography to obtain Tanacetum C;

[0069] The Fr4 was subjected to a third silica gel column chromatography to obtain an eluent containing Tanacetum D;

[0070] The eluent containing Tanacetum D was purified by third-glucan gel column chromatography to obtain Tanacetum D.

[0071] Unless otherwise specified, the raw materials used in this invention are preferably commercially available products.

[0072] This invention involves soaking pyrethrum flowers in an ethanol solution for extraction to obtain a crude extract.

[0073] In this invention, the pyrethrum is preferably the pyrethrum after essential oil extraction. This invention does not specify the method for obtaining the pyrethrum after essential oil extraction; those skilled in the art can proceed using conventional methods for essential oil extraction. This invention uses the pyrethrum after essential oil extraction as raw material to extract the insecticidal active compound described in the above technical solution, resulting in low cost and further improving the utilization rate of pyrethrum.

[0074] In this invention, the volume concentration of the ethanol solution is preferably 70-90%, more preferably 80%. The mass-to-volume ratio of the pyrethrum to the ethanol solution is preferably (15-16) kg:(50-55) L, specifically preferably 15 kg:50 L, 15 kg:55 L, 16 kg:50 L, or 16 kg:55 L. The soaking time is preferably 12-36 h, more preferably 24 h; the soaking temperature is preferably room temperature, i.e., neither additional heating nor additional cooling is required.

[0075] In this invention, the number of extractions is preferably 2 to 4, more preferably 3; the temperature of each extraction is preferably 30 to 80°C, specifically 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C or 80°C, and the time is preferably 10 to 40 minutes, specifically 10 minutes, 20 minutes, 30 minutes or 40 minutes; the extraction method is preferably rotary extraction.

[0076] After extraction, the present invention preferably further includes solid-liquid separation of the obtained extraction system to obtain an extract; and concentration of the extract under reduced pressure to obtain the crude extract. In this invention, the solid-liquid separation is preferably performed by filtration. In this invention, the pressure of the reduced pressure concentration is preferably -0.05 to -0.1 MPa; the temperature of the reduced pressure concentration is preferably 48 to 55°C, specifically preferably 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, or 55°C. The present invention does not specifically limit the time of the reduced pressure concentration, as long as the solvent is removed.

[0077] After obtaining the crude extract, the present invention uses ethyl acetate to extract the crude extract to obtain Tanacetum A-D crude extract.

[0078] In this invention, the preferred mass-to-volume ratio of the crude extract to ethyl acetate is (780–830) g:(5–5.5) L, specifically preferably 780 g:5 L, 790 g:5 L, 800 g:5 L, 810 g:5 L, 820 g:5 L, 830 g:5 L, 780 g:5.5 L, 790 g:5.5 L, 800 g:5.5 L, 810 g:5.5 L, 820 g:5.5 L, or 830 g:5.5 L. In this invention, the extraction is preferably performed 2–4 times.

[0079] Following the extraction, the present invention preferably further includes: removing ethyl acetate from the obtained ethyl acetate phase to obtain the crude Tanacetum A-D extract. In this invention, the method for removing ethyl acetate is preferably vacuum concentration. The present invention does not specifically limit the parameters of the vacuum concentration, as long as the ethyl acetate can be removed.

[0080] After obtaining the crude extract of Tanacetum A-D, the present invention performs silica gel column chromatography elution on the crude extract of Tanacetum A-D, and collects each eluent, which is named Fr1, Fr2, Fr3, Fr4 and Fr5 in sequence; the eluent for the silica gel column chromatography elution is a chloroform-methanol system, and the volume ratio of chloroform to methanol in the chloroform-methanol system is 100:0 to 5:1.

[0081] In this invention, the preferred steps for eluting the crude Tanacetum A-D extract by silica gel column chromatography include: dissolving the crude Tanacetum A-D extract to obtain a solution; mixing the solution with silica gel, then removing the dissolved reagent, packing the column, and performing silica gel column chromatography elution. In this invention, the dissolved reagent is preferably a chloroform-methanol solution, and the volume ratio of chloroform to methanol in the chloroform-methanol solution is preferably 2:1. In this invention, the preferred ratio of the crude Tanacetum A-D extract to the dissolved reagent is 300–600 g: 400–700 mL, specifically 516 g: 650 mL. In this invention, the preferred particle size of the silica gel is 100–300 mesh, specifically 100 mesh, 200 mesh, or 300 mesh. In this invention, the mass ratio of the crude Tanacetum A-D extract to silica gel, based on dry weight, is preferably 1:0.8 to 1.5, specifically preferably 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, or 1:1.5. In this invention, the method for removing dissolved reagents is preferably vacuum concentration. This invention does not specifically limit the method of vacuum concentration, as long as the dissolved reagents can be removed.

[0082] In this invention, during the silica gel column chromatography elution process, the volume ratio of chloroform to methanol in the chloroform-methanol system is preferably 100:0, 100:1, 50:1, 80:1, 10:1, and 5:1, respectively.

[0083] After obtaining Fr2, the present invention performs a first silica gel column chromatography on Fr2 to obtain an eluent containing Tanacetum A-B.

[0084] In this invention, the eluent for the first silica gel column chromatography is preferably a first petroleum ether-ethyl acetate system, wherein the volume ratio of petroleum ether to ethyl acetate in the first petroleum ether-ethyl acetate system is preferably 80:1. Before the first silica gel column chromatography, this invention preferably further includes performing a first purification and a second purification on the silica gel column sequentially. In this invention, the reagent for the first purification is preferably a mixture of petroleum ether and ethyl acetate in a volume ratio of 100:0. In this invention, the reagent for the second purification is preferably a mixture of petroleum ether and ethyl acetate in a volume ratio of 100:1, and the flow rate of the reagent for the second purification is preferably 2-4 mL / min, more preferably 3 mL / min. In this invention, the first purification can remove silica gel impurities; the second purification can remove impurities present in small quantities above the main spot.

[0085] After obtaining the eluent containing Tanacetum A-B, the present invention purifies the eluent containing Tanacetum A-B by first dextran gel column chromatography to obtain the Tanacetum A and the Tanacetum A-B mixture, respectively.

[0086] In this invention, the eluent for the first dextran gel column chromatography purification is preferably methanol. The flow rate of the methanol is preferably 0.4–0.6 mL / min, more preferably 0.5 mL / min. The chromatographic column used for the first dextran gel column chromatography purification is preferably a Sephadex LH-20 gel chromatography column.

[0087] After obtaining the Tanacetum A-B mixture, the present invention performs high performance liquid chromatography elution on the Tanacetum A-B mixture to obtain the Tanacetum B.

[0088] In this invention, the eluent for high-performance liquid chromatography (HPLC) is preferably an acetonitrile-water system, wherein the volume ratio of acetonitrile to water in the acetonitrile-water system is preferably 75:25. In this invention, the flow rate of the acetonitrile-water system is preferably 1–1.5 mL / min, more preferably 1 mL / min.

[0089] After obtaining Fr3, the present invention performs a second silica gel column chromatography on Fr3 to obtain an eluent containing Tanacetum C.

[0090] In this invention, the eluent for the second silica gel column chromatography is preferably a second petroleum ether-ethyl acetate system, wherein the volume ratio of petroleum ether to ethyl acetate in the second petroleum ether-ethyl acetate system is preferably 50:1. Before the second silica gel column chromatography, this invention preferably further includes performing a third, fourth, and fifth impurity removal process on the silica gel column sequentially. In this invention, the reagent for the third impurity removal is preferably a mixture of petroleum ether and ethyl acetate in a volume ratio of 100:0. In this invention, the reagent for the fourth impurity removal is preferably a mixture of petroleum ether and ethyl acetate in a volume ratio of 100:1. In this invention, the reagent for the fifth impurity removal is preferably a mixture of petroleum ether and ethyl acetate in a volume ratio of 80:1. In this invention, the third impurity removal removes silica gel impurities; the fourth impurity removal removes a small amount of impurities above the main spot; and the fifth impurity removal removes a larger amount of impurities above the main spot.

[0091] After obtaining the eluent containing Tanacetum C, the present invention purifies the eluent containing Tanacetum C by second dextran gel column chromatography to obtain the Tanacetum C.

[0092] In this invention, methanol is preferably used as the eluent for the second dextran gel column chromatography purification. The flow rate of the methanol is preferably 0.4–0.6 mL / min, more preferably 0.5 mL / min. The chromatographic column used for the second dextran gel column chromatography purification is preferably a Sephadex LH-20 gel chromatography column.

[0093] After obtaining Fr4, the present invention performs third silica gel column chromatography on Fr4 to obtain an eluent containing Tanacetum D.

[0094] In this invention, the eluent for the third silica gel column chromatography is preferably a third petroleum ether-ethyl acetate system, wherein the volume ratio of petroleum ether to ethyl acetate in the third petroleum ether-ethyl acetate system is preferably 50:1. Before the third silica gel column chromatography, this invention preferably further includes performing a sixth, seventh, and eighth impurity removal process on the silica gel column sequentially. In this invention, the reagent for the sixth impurity removal is preferably a mixture of petroleum ether and ethyl acetate in a volume ratio of 100:0. In this invention, the reagent for the seventh impurity removal is preferably a mixture of petroleum ether and ethyl acetate in a volume ratio of 100:1. In this invention, the reagent for the eighth impurity removal is preferably a mixture of petroleum ether and ethyl acetate in a volume ratio of 80:1. In this invention, the sixth impurity removal can remove silica gel impurities and a small amount of small polar impurities above the main spot; the seventh impurity removal can remove a small amount of impurities above the main spot; and the eighth impurity removal can remove a large amount of impurities above the main spot.

[0095] After obtaining the eluent containing Tanacetum D, the present invention purifies the eluent containing Tanacetum D by third-glucan gel column chromatography to obtain the Tanacetum D.

[0096] In this invention, the eluent for the third dextran gel column chromatography purification is preferably methanol. The flow rate of the methanol is preferably 0.4–0.6 mL / min, more preferably 0.5 mL / min. The chromatographic column used for the third dextran gel column chromatography purification is preferably a Sephadex LH-20 gel chromatography column.

[0097] The present invention also provides the application of the insecticidal active compounds described in the above technical solution in killing aphids.

[0098] In this invention, the aphid is preferably a corn aphid.

[0099] In this invention, when the insecticidal active compound is used to kill aphids, it is preferably used in the form of a drug. The mass percentage of the insecticidal active compound in the drug is preferably 1-99%, more preferably 55-90%. In this invention, the drug preferably also includes pharmaceutical excipients. This invention does not specifically limit the type of pharmaceutical excipients; conventional pharmaceutical excipients in the art can be used.

[0100] In this invention, the dosage form of the drug is preferably a solid dosage form, a liquid dosage form, a semi-solid dosage form, or an aerosol.

[0101] The following detailed description, in conjunction with embodiments, illustrates the insecticidal active compounds, their preparation methods, and applications provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0102] Example 1

[0103] Extract Tanacetum A-B, a compound with good insecticidal activity.

[0104] 1. 15 kg of pyrethrum flowers after essential oil extraction were soaked in 80% ethanol for 24 hours. The soaking mixture was then subjected to constant-temperature rotary extraction, filtration, and vacuum concentration to remove the solvent, yielding 815 g of crude extract of secondary metabolites. The mass-to-volume ratio of 80% ethanol to pyrethrum flowers after essential oil extraction was 15 kg: 50 L. The rotary extraction temperature was 55℃, with each extraction lasting 30 minutes, repeated 3 times. Vacuum concentration was performed at 55℃ and a pressure of -0.05 to -0.1 MPa.

[0105] 2. The crude extract of secondary metabolites was extracted with ethyl acetate. The ethyl acetate phase obtained from the extraction was concentrated under reduced pressure to remove the ethyl acetate, yielding 516 g of crude Tanacetum A-D extract. The mass-to-volume ratio of the crude alcohol extract of secondary metabolites to ethyl acetate was 800 g: 5.5 L, and the extraction was performed three times.

[0106] 3. Dissolve 516g of crude Tanacetum A-D extract in 650mL of chloroform-methanol solution with a volume ratio of 2:1, then mix with 520g of silica gel (100 mesh), concentrate under reduced pressure to remove solvent, and pack into a column; perform gradient elution sequentially with chloroform:methanol volume ratios of 100:0, 100:1, 50:1, 80:1, 10:1, and 5:1, and combine the elution fractions to obtain 5 fractions, named Fr1 to Fr5 respectively.

[0107] 4. The Fr2 fraction was subjected to silica gel column chromatography. First, 300 mL of petroleum ether:ethyl acetate (volume ratio 100:0) was used to elute to remove silica gel impurities. Then, 300 mL of petroleum ether:ethyl acetate (volume ratio 100:1) was used to elute to remove a small amount of impurities above the main spot. Finally, about 200 mL of petroleum ether:ethyl acetate (volume ratio 80:1) was used to elute until the target compound began to elute. The eluent was continued to be washed with petroleum ether:ethyl acetate (volume ratio 80:1) until the target compound disappeared. The eluents containing the relatively pure target compound were combined to obtain the eluent containing Tanacetum A-B.

[0108] 5. The eluent was purified by dextran gel column chromatography using methanol (flow rate 0.5 mL / min) as solvent and dried to obtain a mixture of 70.4 mg Tanacetum A and 20.3 mg Tanacetum A-B.

[0109] 6. The 20.3 mg Tanacetum A-B mixture was eluted by HPLC with gradient elution (eluting agent: acetonitrile:water = 75:25, flow rate: 1 mL / min), and dried to obtain 2.3 mg Tanacetum B.

[0110] Structural identification

[0111] The compounds Tanacetum A and Tanacetum B, and their structures, were identified by 1D / 2D NMR (one-dimensional nuclear magnetic resonance spectroscopy and two-dimensional nuclear magnetic resonance spectroscopy) and HR-ESI-MS (high-resolution electrospray ionization mass spectrometry).

[0112] Structural identification of Tanacetum A

[0113] The HR-ESI-MS spectrum of Tanacetum A can be found in [link to HR-ESI-MS spectrum]. Figure 1 ,like Figure 1 As shown: The molecular formula of this compound is C2. 25 H 24 O8 (m / z: 453.1542 [M+H]) + The calculated value is 453.1544, and the degree of unsaturation is 14.

[0114] The results of 1D / 2D NMR detection of Tanacetum A are as follows: Figures 2-5 As shown, the TanacetumA compound is based on 1 H-NMR, 13C-NMR and DEPT spectral data indicate δC (ppm): two methyl groups at 20.18 and 27.39, four methylene groups at 61.19, 70.39, 101.19, and 102.05, ten methine groups at 47.34, 50.83, 83.58, 106.48, 108.00, 108.11, 108.33, 115.22, 119.65, and 124.66, and nine quaternary carbons at 132.41, 135.25, 147.39, 148.08, 148.44, 152.12, 157.45, 165.86, and 196.9. 1 H- 1 1H COSY spectroscopy revealed the C-7`-C-8`-C-8-C-9` fragment with correlation signals of H-7` / H-8` / H-9` and H-8 / H-8`H-9`, supported by HSQC. HMBC plots showed correlations between H2-10` and C-4`, C-3`, H2-10 and C-4, C-3, H-7` and C-6`, C-2`, C-9`, C-6 and C-2, H-8 and C-7`, C-9`, and C-7, and H2-9` and C-11. This revealed two oxygen ring fragments, C-10`-C-4`-C-3` and C-10-C-4-C-3, and a tetrahydrofuran fragment, C-7`-C-8`-C-8-C-9, thus confirming the carbon skeleton of compound Tanacetum A. The correlation between H-7` / H-9` and H-8` / H9` in the NOESY spectrum indicates that C-13 and C-12 have an E configuration, with H-7`, H-8`, and H-9` on the same side, while H-7` / H-8 is not correlated and is on opposite sides. Therefore, the relative configuration of compound Tanacetum A is determined to be 7`R,8`R,8S. A conformational search was performed on the two possible conformations of Tanacetum A, 7`R,8`R,8S and 7`S,8`S,8R. For conformations with a distribution ratio greater than 1%, density functional theory (DFT) was used to perform ECD calculations at the B3LYP / 6-311+G(d,p) level using Gauss 09. By comparing the results with the experimental ECD spectrum, the absolute configuration of compound Tanacetum A was determined to be 7`R,8`R,8S.

[0115] In summary, Tanacetum A has the following structure:

[0116]

[0117] It belongs to the lignan class of compounds, and its chemical name is: (7`R,8`R,8S)-(2-(benzo[d][1,3]dioxol-5-yl)-4-(benzo[d][1,3]dioxole-5-carbonyl)tetrahydrofuran-3-yl)methyl 3-methylbut-2-enoate; that is: ((7`R,8`R,8S)-2-(benzo[d][1,3]dioxol-5-yl)-4-(benzo[d][1,3]dioxole-5-carbonyl)tetrahydrofuran-3-yl)methyl 3-methylbut-2-enoate.

[0118] Structural identification of Tanacetum B

[0119] The results of 1D / 2D NMR detection of Tanacetum B are as follows: Figures 6-10 As shown, the TanacetumB compound is based on 1 H-NMR, 13 C-NMR and DEPT spectral data indicate δC (ppm): two methyl groups at 20.39 and 27.56, four methylene groups at 61.84, 71.08, 101.21, and 102.13, ten methine groups at 49.91, 50.04, 84.08, 107.23, 108.12, 108.20, 108.38, 115.43, 120.53, and 124.89, and nine quaternary carbons at 131.48, 134.12, 147.61, 148.08, 148.46, 152.26, 157.97, 166.4, and 196.91. 1 H- 11H COSY spectroscopy revealed the C-7`-C-8`-C-8-C-9` fragment with correlation signals of H-7` / H-8` / H-9` and H-8 / H-8`H-9`, supported by HSQC. HMBC plots showed correlations between H2-10` and C-4`, C-3`, H2-10 and C-4, C-3, H-7` and C-6`, C-2`, C-9`, C-6 and C-2, H-8 and C-7`, C-9`, and C-7, and H2-9` and C-11. This revealed two oxygen ring fragments, C-10`-C-4`-C-3` and C-10-C-4-C-3, and a tetrahydrofuran fragment, C-7`-C-8`-C-8-C-9, thus confirming the carbon skeleton of compound Tanacetum B. The correlation between H-7` / H-9` / H-8 and H-8` / H-9` / H-9 in the NOESY spectrum indicates that C-13 and C-12 have an E configuration, with H-7`, H-8`, H-9`, and H-8 on the same side. Therefore, the relative configuration of compound Tanacetum B was determined to be 7`R,8`R,8R. Conformation searches were performed on the two possible conformations of compound Tanacetum B, 7`R,8`R,8R and 7`S,8`S,8S. For conformations with a distribution ratio greater than 1%, density functional theory (DFT) was used to perform ECD calculations at the B3LYP / 6-311+G(d,p) level using Gauss 09. By comparing with the experimental ECD spectrum, the results showed that the absolute configuration of compound Tanacetum B was determined to be 7`S,8`S,8S.

[0120] In summary, the compound Tanacetum B has the following structure:

[0121]

[0122] It belongs to the lignan class of compounds, and its chemical name is ((7′S,8′S,8S)-2-(benzo[d][1,3]dioxol-5-yl)-4-(benzo[d][1,3]dioxole-5-carbonyl)tetrahydrofuran-3-yl)methyl 3-methylbut-2-enoate, which is ((7′S,8′S,8S)-2-(benzo[d][1,3]dioxol-5-yl)-4-(benzo[d][1,3]dioxol-5-carbonyl)tetrahydrofuran-3-yl)3-methylbut-2-enoate.

[0123] The chemical shift δ of compounds Tanacetum A and Tanacetum B is assigned as shown in Table 1.

[0124] Table 1. Assignment of chemical shift δ for compounds Tanacetum A and Tanacetum B

[0125]

[0126] Example 2: Extraction of Tanacetum C, a compound with good insecticidal activity.

[0127] Examples 1, 2, and 3 are the same as in Example 1.

[0128] 4. The Fr3 fraction was subjected to silica gel column chromatography. First, 300 mL of petroleum ether:ethyl acetate (volume ratio 100:0) was used to elute and remove silica gel impurities. Then, 300 mL of petroleum ether:ethyl acetate (volume ratio 100:1) was used to elute and remove a small amount of impurities above the main spot. Next, about 200 mL of petroleum ether:ethyl acetate (volume ratio 80:1) was used to elute and remove a larger amount of impurities above the main spot. Finally, petroleum ether:ethyl acetate (volume ratio 50:1) was used to wash until the target compound began to elute. Washing was continued with petroleum ether:ethyl acetate (volume ratio 50:1) until the target compound disappeared. The eluents containing the relatively pure target compound were combined to obtain the eluent containing Tanacetum C.

[0129] 5. Using methanol (flow rate 0.5 mL / min) as solvent, the eluent was purified by dextran gel column chromatography (Sephadax LH-20 gel chromatography column), dried, and 8.7 mg Tanacetum C was obtained.

[0130] Tanacetum C structural identification

[0131] The compound Tanacetum C and its structure were identified by 1D / 2D NMR (one-dimensional nuclear magnetic resonance spectroscopy and two-dimensional nuclear magnetic resonance spectroscopy) and HR-ESI-MS (high-resolution electrospray ionization mass spectrometry).

[0132] The HR-ESI-MS spectrum of Tanacetum C can be found in [link to HR-ESI-MS spectrum]. Figure 11 ,like Figure 11 As shown, the molecular formula of this compound is C2. 11 H 16 O3(m / z:C 11 H 16 NaO3: 219.0992 (calculated value: 219.0997), with an unsaturation degree of 4.

[0133] The results of 1D / 2D NMR detection are as follows Figures 12-15 As shown, based on 1 H-NMR, 13C-NMR and DEPT spectral data indicate 13.97 and 22.82 methyl groups at δC (ppm); 22.0 and 44.52 methylene groups at δC (ppm); and 62.9, 71.4, 126.3, and 135.74 methine groups at δC (ppm). 1H-1H COSY spectroscopy, supported by correlation signals from H-4 / H-5 / H-3 and H-8 / H-9 and HSQC, reveals a C-3-C-5 fragment. HMBC plots show correlations between H-9 and C-6, C-7, C-8, and C-10; H2-5 and C-3, C-10 and C-7; H-4 and C-2, C-6; and H3-1 and C-3, revealing a C-6-C-10 five-membered ring fragment and a C-1-C-6 chain fragment, thus defining the carbon skeleton of compound 2. The correlation between H-3 / H-4 and H-9 / H3-11 in the NOESY spectrum indicates that C-3 and C-4 have a Z configuration, with H-9 and H3-11 on the same side, while H-2 / H-4 is not correlated and is on opposite sides. A conformational search was performed on the four possible conformations of compound Tanacetum C: 2R,9R, 2S,9S, 2R,9S, and 2S,9R. For conformations with a distribution ratio greater than 1%, density functional theory (DFT) was used to perform ECD calculations at the B3LYP / 6-311+G(d,p) level using Gauss 09. Comparison with the experimental ECD spectrum results indicates that the absolute configuration of compound Tanacetum C is 2S,9R or 2S,9S. To determine the relative configuration of the C-9 position, the GIAO method was used at the PCM / Mpw1pw91 / 6311+G(d,p) level to analyze two reasonable configurations labeled 2S,9R or 2S,9S. 1 H and 13 3C NMR chemical shift calculations were performed using TAD, MAE, and DP4. + Probabilistic analysis was performed to comprehensively compare the theoretical chemical shifts of 2S,9R or 2S,9S with experimental values, and the results are shown in Table 2. The results indicate that 2S,9R is the most likely configuration.

[0134]

[0135] Table 2 DP4 of 2S,9R or 2S,9S + Theoretical chemical shift obtained from probabilistic analysis and experimental values

[0136] <![CDATA[ 1 H]]> 95.10％ <![CDATA[ 1 H]]> 0.00％ <![CDATA[ 13 C]]> 62.01％ <![CDATA[ 13 C]]> 22.84％ <![CDATA[ 1 H+ 13 C]]> 99.30％ <![CDATA[ 1 H+ 13 C]]> 0.00％

[0137] In summary, the structural formula of the compound Tanacetum C prepared in Example 2 can be determined as follows:

[0138]

[0139] It is evident that compound Cetacean C belongs to the monoterpene ester class, enriching the diversity of monoterpene ester compounds. Its chemical name is (4R)-4-hydroxy-2-((E)-4-hydroxypent-2-en-1-yl)-3-methylcyclopent-2-en-1-one, which is (4R)-4-hydroxy-2-((E)-4-hydroxypent-2-en-1-yl)-3-methylcyclopent-2-en-1-one.

[0140] Example 3

[0141] Preparation of the compound Tanacetum D with good insecticidal activity

[0142] Examples 1, 2, and 3 are the same as in Example 1.

[0143] 4. The Fr4 fraction was subjected to silica gel column chromatography. First, 250 mL of petroleum ether:ethyl acetate (volume ratio 100:0) was used to elute to remove silica gel impurities and a small amount of small polar impurities above the main spot. Then, 250 mL of petroleum ether:ethyl acetate (volume ratio 100:1) was used to elute to remove a small amount of impurities above the main spot. Then, about 300 mL of petroleum ether:ethyl acetate (volume ratio 80:1) was used to elute to remove a large amount of impurities above the main spot. Finally, petroleum ether:ethyl acetate (volume ratio 50:1) was used to wash until the target compound began to elute. Washing was continued with petroleum ether:ethyl acetate (volume ratio 50:1) until the target compound disappeared. The eluents containing the relatively pure target compound were combined to obtain the eluent containing Tanacetum D.

[0144] 5. Using methanol as solvent, the eluent was purified twice by dextran gel column chromatography (Sephadax LH-20 gel chromatography column), dried, and 11.3 mg Tanacetum D was obtained.

[0145] TanacetumD structural identification

[0146] The compound Tanacetum D and its structure were identified by 1D / 2D NMR (one-dimensional nuclear magnetic resonance spectroscopy and two-dimensional nuclear magnetic resonance spectroscopy) and HR-ESI-MS (high-resolution electrospray ionization mass spectrometry).

[0147] The HR-ESI-MS spectrum of Tanacetum D can be found in [link to HR-ESI-MS spectrum]. Figure 16 ,like Figure 16 As shown, the molecular formula of this compound is C2. 17 H28O5(m / z:C 17 H 28 NaO5: 335.1826 (calculated value: 335.1829), with an unsaturation degree of 4.

[0148] The results of 1D / 2D NMR detection are as follows Figures 17-20 shown. Based on 1 1H-NMR spectrum analysis shows that δH(ppm) 2.06, S, 4.61, S indicates the presence of an acetyl group structure. 13 13C-NMR and DEPT data indicate that there are 8 methylene groups at δC(ppm) 112.0, 42.0, 22.6, 31.4, 34.1, 64.0, 112.5, 61.9; 2 methyl groups at δC(ppm) 21.0, 22.58; and 3 methine groups at δC(ppm) 144.8, 131.6, 73.8. Through 1 1H- 1 1H COSY spectrum shows that the correlation signals of H2-5 / H2-4 / H-6, H2-9 / H2-8 / H-10, H2-1 / H1-2, and with the assistance of the correlation of HSQC, reveal the chain fragments of C-4-C-6, C-8-C-10 and the alkenyl fragment of C-1-C-2. The HMBC spectrum shows that H-6 is related to C-8, C-4, C-5, C-7, H2-4 is related to C-2, C-3, C-5, H2-14 is related to C-6, C-8, C-7, and H-10 is related to C-8, C-12, C-9, C-11, so as to determine the carbon skeleton of Compound 1. The correlations between H2-1 / H-2, H-6 / H2-14, H2-8 / H2-14, H2-4 / H-6 in the NOESY spectrum indicate that C-1 and C-2 are in the Z configuration, H-6, H2-14, H2-8, H2-4 are on the same side, and H-10 / H2-12 has no correlation and is on the opposite side.

[0149] In summary, it can be determined that the structural formula of the compound Tanacetum D prepared in Example 3 is:

[0150]

[0151] As can be seen from the above formula: Compound Tanacetum D belongs to sesquiterpenoid compounds, and its chemical name is: (Z)-6-hydroxy-2-((R)-3-hydroxy-4-(hydroxymethyl)pent-4-en-1-yl)-6-methylocta-2,7-dien-1-yl acetate, that is: (Z)-6-hydroxy-2-((R)-3-hydroxy-4-(hydroxymethyl)pent-4-en-1-yl)-6-methylocta-2,7-dien-1-yl acetate.

[0152] The chemical shift δ assignments of Compound Tanacetum C and Tanacetum D are shown in Table 3.

[0153] Table 3. Assignment of chemical shift δ for compounds Tanacetum C and Tanacetum D.

[0154]

[0155]

[0156] Insecticidal activity of compound Tanacetum A-D

[0157] Aphids, a collective term for insects in the superfamily Aphidoidea of ​​the order Hemiptera, commonly congregate on seedlings, tender leaves, young stems, and leaves near the ground, feeding on the sap of their hosts. Aphids can reproduce 20-30 generations per year, and offspring can reproduce within just 5 days of hatching. They feed by piercing and sucking plant sap, causing serious damage to crops, fruit trees, and forests. The following are some of the main aspects of aphid damage:

[0158] Direct damage: Aphids suck sap from plants using their piercing-sucking mouthparts, causing leaves to lose their green color, fall off prematurely, or branches to wither. Severely affected plants may experience stunted growth, reduced yield, or even death.

[0159] Spreading viruses: Aphids are vectors for many plant viruses. While sucking plant sap, they can transmit viruses to healthy plants, causing viral diseases such as cucumber mosaic virus (CMV) and resulting in greater economic losses.

[0160] Inducing sooty mold: When aphids damage plants, they secrete a lot of honeydew. This honeydew contaminates the branches and leaves, providing a good growth environment for fungi, leading to sooty mold, affecting the plant's photosynthesis, and further weakening the plant's growth.

[0161] Impact on ornamental value: For ornamental plants, aphid damage not only affects plant growth, but also causes leaves to curl and wrinkle, flower buds to wither, flowering period to be shortened, and flower color to be diminished, seriously reducing ornamental value.

[0162] The following method was adopted: Spraying method, Part 9 of the Ministry of Agriculture and Rural Affairs' industry standard for indoor bioassay of pesticides.

[0163] (1) Select corn aphids that have been continuously raised indoors and have the same physiological state.

[0164] (2) The compounds Tanacetum A-D and the positive control drug imidacloprid were prepared into a stock solution of 1 mg / mL using the organic solvent acetone. Then, five series of mass gradients of 100 mg / L, 60 mg / L, 30 mg / L, 10 mg / L and 3.3 mg / L were prepared using 0.1% Tween-80 aqueous solution and dispensed into five 5 mL small spray bottles.

[0165] (3) Make many small holes in the petri dish with an iron needle to facilitate air circulation. Place a round cabbage leaf of similar size in the petri dish. Use a paintbrush to select 20 corn aphids of the same physiological state and place them in the petri dish. Perform quantitative spraying on both the front and back sides, with a spray volume of 1 mL. Each treatment is repeated 4 times, and a blank control is set up with no pesticide (containing all solvents and emulsifiers). Imidacloprid is set as a positive control.

[0166] (4) The treated corn aphids were raised and observed under the conditions of (25±1)℃, 60%~80% relative humidity and L:D(16:8)h photoperiod.

[0167] (5) Record the mortality of corn aphids 24 hours after treatment, including the total number of insects and the number of dead insects.

[0168] (6) Data statistics and analysis: Calculate the corrected mortality rate for each treatment based on the survey data.

[0169] P = K / N × 100% (Formula 1)

[0170] In Formula 1, P represents the mortality rate as a percentage (%); K represents the number of dead insects as heads; and N represents the total number of insects treated as heads.

[0171] P1=(P t -P0) / (1-P0)×100% Formula 2

[0172] In Formula 2, P1 is the corrected mortality rate, expressed as a percentage (%); P t -- Treatment mortality rate, in percentage (%); P0 -- Blank control mortality rate, in percentage (%).

[0173] If the control mortality rate is <5%, no correction is needed; if the control mortality rate is between 5% and 20%, correction should be performed according to Formula 2; if the control mortality rate is >20%, the trial needs to be repeated.

[0174] (7) The data were processed using probability value analysis and analyzed using GraphPadPrism 9 software to calculate the lethal concentration LC50 value.

[0175] The LC50 values ​​of the insecticidal activity of compounds Tanacetum A-D were 6.856 mg / L, 7.932 mg / L, 33.160 mg / L, and 9.311 mg / L, respectively, while the LC50 value of the positive control imidacloprid was 25.856 mg / L. Compound Tanacetum A-D exhibited significant insecticidal activity against corn aphids, which is of great significance for the development of novel biological insecticides. The method of producing large quantities of the insecticidal compound Tanacetum A-D using pyrethrum extraction is simple and effective, reflecting the needs of modern environmental protection and a low-carbon economy, and realizing the reuse of waste. Furthermore, it lays a solid foundation for further research and industrial development of this insecticide, and has broad market application prospects.

[0176] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An insecticidal active compound, characterized in that, It has the following structure:

2. The method for preparing the insecticidal active compound according to claim 1, characterized in that, Includes the following steps: Pyrethrum flowers were soaked in an ethanol solution for extraction to obtain a crude extract. The crude extract was extracted with ethyl acetate to obtain crude extracts of Tanacetum A-D. The crude extracts of Tanacetum A-D were eluted by silica gel column chromatography, and the eluents were collected and named Fr1, Fr2, Fr3, Fr4 and Fr5 in sequence. The eluent for the silica gel column chromatography was a chloroform-methanol system, in which the volume ratio of chloroform to methanol was 100:0 to 5:

1. The Fr2 was subjected to a first silica gel column chromatography to obtain an eluent containing Tanacetum A-B; The eluent containing Tanacetum A-B was purified by first dextran gel column chromatography to obtain Tanacetum A and the mixture of Tanacetum A-B, respectively. The Tanacetum A-B mixture was eluted by high performance liquid chromatography to obtain Tanacetum B; The Fr3 was subjected to a second silica gel column chromatography to obtain an eluent containing Tanacetum C; The eluent containing Tanacetum C was purified by second dextran gel column chromatography to obtain Tanacetum C; The Fr4 was subjected to a third silica gel column chromatography to obtain an eluent containing Tanacetum D; The eluent containing Tanacetum D was purified by third-glucan gel column chromatography to obtain Tanacetum D.

3. The preparation method according to claim 2, characterized in that, The pyrethrum mentioned is the pyrethrum after essential oil extraction; The volume concentration of the ethanol solution is 70-90%, and the mass-to-volume ratio of the pyrethrum to the ethanol solution is (15-16) kg: (50-55) L; the soaking time is 12-36 h. The extraction is performed 2 to 4 times, with each extraction at a temperature of 30 to 80°C and a time of 10 to 40 minutes. After extraction, the process further includes solid-liquid separation of the obtained extraction system to obtain an extract; and concentration of the extract under reduced pressure to obtain the crude extract.

4. The preparation method according to claim 2, characterized in that, The mass-to-volume ratio of the crude extract to ethyl acetate is (780–830) g: (5–5.5) L.

5. The preparation method according to claim 2, characterized in that, During the silica gel column chromatography elution process, the volume ratio of chloroform to methanol in the chloroform-methanol system is 100:0, 100:1, 50:1, 80:1, 10:1, and 5:1, respectively.

6. The preparation method according to claim 2, characterized in that, The eluent for the first silica gel column chromatography is a first petroleum ether-ethyl acetate system, wherein the volume ratio of petroleum ether to ethyl acetate in the first petroleum ether-ethyl acetate system is 80:

1.

7. The preparation method according to claim 2 or 6, characterized in that, The eluent for the first dextran gel column chromatography purification was methanol; The eluent used in the high-performance liquid chromatography is an acetonitrile-water system, wherein the volume ratio of acetonitrile to water in the acetonitrile-water system is 75:

25.

8. The preparation method according to claim 2, characterized in that, The eluent for the second silica gel column chromatography is a second petroleum ether-ethyl acetate system, wherein the volume ratio of petroleum ether to ethyl acetate in the second petroleum ether-ethyl acetate system is 50:

1. The eluent for the second dextran gel column chromatography purification was methanol.

9. The preparation method according to claim 2, characterized in that, The eluent for the third silica gel column chromatography is a third petroleum ether-ethyl acetate system, wherein the volume ratio of petroleum ether to ethyl acetate in the third petroleum ether-ethyl acetate system is 50:

1. The eluent used for the third dextran gel column chromatography purification was methanol.

10. The use of the insecticidal active compound of claim 1 in killing aphids.

Images