Extraction method of gelsemium alkaloid monomer and application thereof in crop disease and pest control

Abstract

The application discloses a kind of gelsemium alkaloid monomer extraction method and its application in crop disease and pest control, belong to biological pesticide technical field.The gelsemium alkaloid monomer extraction method of the application includes the following steps: adding sodium hydroxide solution and ethanol solution in gelsemium plant tissue, obtain extraction liquid;Extracted liquid is concentrated by rotary evaporation, then extracted with dichloromethane, collect extraction liquid, recover solvent under reduced pressure and dry, to obtain crude extract;Crude extract is separated and purified by preparative high performance liquid chromatograph, to obtain the gelsemium alkaloid monomer.The method is fast, and the product has high purity;And the gelsemium alkaloid monomer obtained as biological source pesticide shows high biological activity, can effectively prevent and control crop disease and pest;Especially when it is prepared into nano biological pesticide, it shows higher disease and pest control effect, and has wide application prospect in agricultural field.

Description

Technical Field

[0001] This invention relates to a method for extracting Gelsemium alkaloid monomers and their application in the control of crop diseases and pests, belonging to the field of nanopesticide technology. Background Technology

[0002] Gelsemium elegans Benth., an evergreen woody vine belonging to the genus Gelsemium in the family Loganiaceae, is a traditional Chinese medicine with strong biotoxicity. Commonly known as "heartbreak grass" or "big tea medicine," it contains various structurally specific alkaloids, which have been shown to possess pharmacological activities such as immunomodulation, anti-tumor effects, and treatment of psoriasis and neurodermatitis. Furthermore, Gelsemium alkaloids exhibit neuroprotective, cognitive-enhancing, analgesic, and anti-anxiety effects, with the anti-anxiety effect being particularly prominent. Current research on Gelsemium primarily focuses on veterinary medicine, with limited reports on its application in agricultural pest and disease control.

[0003] Current methods for preparing Gelsemium alkaloid monomers primarily rely on high-speed countercurrent chromatography (HSCCC). While HSCCC is suitable for large-scale separation with sample loading up to the gram level, its separation efficiency for structurally similar alkaloid monomers is unsatisfactory, resulting in low product purity. Furthermore, this method is costly and requires strict operational procedures. Overall, the current separation and preparation of Gelsemium alkaloids still suffers from cumbersome processes, high costs, low separation efficiency, and insufficient monomer purity. Therefore, it is necessary to establish an efficient, stable, and convenient extraction and separation system to support subsequent research on the active substances in Gelsemium elegans.

[0004] Biopesticides are a rapidly developing alternative to chemical pesticides in recent years, possessing advantages such as low toxicity, easy degradation, and environmental friendliness. They help alleviate environmental pollution problems caused by chemical pesticides in crop pest control. However, biopesticides are susceptible to environmental factors such as high temperature and strong sunlight during field application, leading to unstable efficacy. Therefore, using nano-delivery systems to load biopesticides can improve their environmental stability, thereby enhancing their control of crop diseases and demonstrating practical value. Summary of the Invention

[0005] [Technical Issues] Existing gelseminine alkaloid preparation technologies still suffer from problems such as complex extraction processes, high costs, poor separation effects, and low purity of obtained monomers; furthermore, research on the application of gelseminine alkaloids in the agricultural field is limited.

[0006] [Technical Solution] To address the problems existing in the prior art, the purpose of this invention is to provide an efficient and convenient extraction technology for Gelsemium alkaloid monomers. This technology offers fast extraction speed and high product purity. Furthermore, the obtained Gelsemium alkaloid monomers exhibit high biological activity when used as biopesticides, effectively preventing crop diseases and pests. In particular, when prepared into nano-biopesticides, they demonstrate even higher disease and pest control effects, and have broad application value in agriculture.

[0007] To achieve the above objectives, the following technical solution is provided: The first objective of this invention is to provide a method for extracting monomers of Gelsemium elegans alkaloids, the method comprising the following steps: (1) Add sodium hydroxide solution to the Gelsemium sample, stir well and let stand, then add ethanol solution, extract by ultrasonication, and collect the supernatant; repeat 2-3 times, combine the supernatants, and the extract is the extract; (2) The extract from step (1) is concentrated by rotary evaporation, and the pH of the concentrate is adjusted to 2-3. After standing, it is filtered. The filtrate is extracted with dichloromethane to remove the organic phase. Then the pH of the filtrate is adjusted to 10-11 and allowed to stand. Then it is extracted with dichloromethane again, the extract is collected, the solvent is recovered under reduced pressure, and it is dried to obtain the crude extract. (3) Dissolve the crude extract powder obtained in step (3) in acetonitrile and filter it through an organic filter membrane. The filtrate is separated and purified by preparative high performance liquid chromatography (HPLC) to obtain gelseminale A and gelseminale spores respectively.

[0008] In one embodiment, the Gelsemium sample in step (1) is a pretreated Gelsemium sample powder.

[0009] In one embodiment, the preparation of the Gelsemium sample powder specifically involves cutting the Gelsemium into small segments, drying them to a constant weight, and then pulverizing them using a pulverizer.

[0010] In one embodiment, the drying refers to drying in a forced-air drying oven at 60~70°C.

[0011] In one embodiment, the sodium hydroxide solution in step (1) has a mass fraction of 3-5%.

[0012] In one embodiment, the ratio of the gelsemium sample to the sodium hydroxide solution in step (1) is 1~3:10~15g:mL.

[0013] In one embodiment, the ethanol solution in step (1) has a mass fraction of 75%.

[0014] In one embodiment, the amount of ethanol solution added in step (1) is 10 to 15 times the mass of the Gelsemium elegans sample.

[0015] In one embodiment, before the rotary evaporation concentration in step (2), the extract can be placed in a Buchner funnel for filtration to remove the precipitate in the extract and obtain a clear extract.

[0016] In one embodiment, the filter paper diameter of the Buchner funnel is 80-100 mm.

[0017] In one embodiment, the rotary evaporation concentration in step (2) is stopped when the volume is reduced to 1 / 10 of the initial volume or when there is no obvious ethanol odor.

[0018] In one embodiment, the settling time in step (2) is 20 to 30 minutes.

[0019] In one embodiment, the detection conditions of the preparative high-performance liquid chromatograph in step (3) are as follows: column: YMC-PackProC18 (250 mm × 20 mm, 5 μm) column; column temperature: 30 ℃; detection wavelength: 254 nm; injection volume: 300 µL; mobile phase: phase A: 0.5% formic acid aqueous solution, phase B: acetonitrile, elution program: 0~4 min, 10%~10%B; 4~14 min, 10%~14%B; 14~25 min, 14%~24%B; 25~35 min, 24%~24%B; 35~40 min, 24%~90%B; 40~60 min, 90%~10%B.

[0020] In one embodiment, the specific process of separation and purification in step (3) is as follows: the eluent of gelsemium elegans and gelsemium methyl is collected in sections, the product peaks of gelsemium methyl and gelsemium elegans are separated, the regions with higher purity are collected directly, and the mixed peaks containing the two products are prepared a second time; the post-processing of the prepared products is to remove the solvent by vacuum distillation, and after the solvent is evaporated by rotary evaporation, the sample is placed in a forced-air drying oven and dried to constant weight, thus successfully preparing the gelsemium methyl and gelsemium elegans samples.

[0021] In one embodiment, the extraction rate of gelsemin A is 8.4%, and the extraction rate of gelseminoid is 7.2%.

[0022] A second objective of this invention is to provide gelseminine A and gelseminine ions obtained by the extraction method described above.

[0023] In one embodiment, the structures of gelseminine A and gelseminine B are shown in Formulas I and II: Formula I; Formula II.

[0024] In one embodiment, the purity of gelsemin A is 97.1%, and the purity of gelseminoid is 98.4%.

[0025] The third objective of this invention is to provide an application of the aforementioned gelseminine A and gelseminine seeds in the prevention and control of crop diseases and pests.

[0026] A fourth objective of this invention is to provide a method for preparing gelsemine@ZIF-8 nanoparticles, the method comprising the following steps: 2-Methylimidazole was dissolved in water, and gelsemine A obtained by the above method was added. The mixture was magnetically stirred at room temperature to obtain a 2-methylimidazole solution. Then, Zn(NO3)2·6H2O was dissolved in water and slowly added dropwise to the 2-methylimidazole solution under stirring. The reaction was carried out at room temperature. After the reaction was completed, the mixture was centrifuged and the precipitate was washed to obtain Gelsemine@ZIF-8 nanoparticles.

[0027] In one embodiment, the mass ratio of 2-methylimidazole, gelsminoid A, and water is 6~8:0.05:40~50.

[0028] In one embodiment, the mass ratio of Zn(NO3)2·6H2O to 2-methylimidazole is 0.1~0.5:2~8.

[0029] In one embodiment, the reaction time is 30-60 minutes.

[0030] In one embodiment, the centrifugation conditions are 10,000 to 15,000 rpm and the time is 10 to 15 minutes.

[0031] In one embodiment, the washing process involves washing with deionized water and ethanol 2 to 3 times each.

[0032] The fifth objective of this invention is to provide Gelsemine@ZIF-8 nanoparticles prepared by the method described above.

[0033] In one embodiment, the average size of the Gelsemine@ZIF-8 nanoparticles is 240~280 nm.

[0034] The sixth objective of this invention is to provide a method for preparing Koumine@ZIF-8 nanoparticles, the method comprising the following: 2-Methylimidazole was dissolved in water, and gelsemium oleoresin obtained by the above method was added. The mixture was magnetically stirred at room temperature to obtain a 2-methylimidazole solution. Then, Zn(NO3)2·6H2O was dissolved in water and slowly added dropwise to the 2-methylimidazole solution under stirring. The reaction was carried out at room temperature. After the reaction was completed, the mixture was centrifuged and the precipitate was washed to obtain Koumine@ZIF-8 nanoparticles.

[0035] In one embodiment, the mass ratio of 2-methylimidazole, gelsemium elegans, and water is 6-8:0.05:40-50.

[0036] In one embodiment, the mass ratio of Zn(NO3)2·6H2O to 2-methylimidazole is 0.1~0.5:2~8.

[0037] In one embodiment, the reaction time is 30-60 minutes.

[0038] In one embodiment, the centrifugation conditions are 10,000 to 15,000 rpm and the time is 10 to 15 minutes.

[0039] The seventh object of the present invention is to provide Koumine@ZIF-8 nanoparticles prepared by the method described above.

[0040] In one embodiment, the Koumine@ZIF-8 nanoparticles have an average size of 250~310 nm.

[0041] The eighth objective of this invention is to provide an application of the above-described Koumine@ZIF-8 nanoparticles or Gelsemine@ZIF-8 nanoparticles in the prevention and control of crop diseases and pests.

[0042] A ninth objective of the present invention is to provide a botrytis cinerea inhibitor comprising the active ingredient gelseminine A and / or gelseminine ions.

[0043] In one embodiment, the gelsemine A comprises gelsemine A monomer or gelsemine A nanoparticles (Gelsemine@ZIF-8 nanoparticles).

[0044] In one embodiment, the gelseminates comprise gelseminate monomers or gelseminate nanoparticles (Koumine@ZIF-8 nanoparticles).

[0045] In one embodiment, the concentration of gelseminine A or gelseminine is 50~200 mg / L.

[0046] The tenth objective of this invention is to provide an application of gelseminine alkaloids in the control of tobacco aphids, wherein the application involves preparing a solution of gelseminine alkaloids and spraying it onto tobacco leaves.

[0047] In one embodiment, the gelseminine alkaloids include one or more of gelseminine A monomer, gelsemine@ZIF-8 nanoparticles, gelseminine A monomer, and Koumine@ZIF-8 nanoparticles.

[0048] In one embodiment, the concentration of the gelsemium alkaloid solution is 50~200 mg / L.

[0049] In one embodiment, the spraying is performed during the seedling stage, the root extension stage (grouping stage), or the vigorous growth stage; preferably, during the seedling stage.

[0050] The eleventh objective of this invention is to provide a method for improving the control effect of tobacco aphids, wherein the method involves preparing a solution of Gelsemine@ZIF-8 nanoparticles or Koumine@ZIF-8 nanoparticles and spraying it onto tobacco leaves.

[0051] In one embodiment, the concentration of the solution of the Gelsemine@ZIF-8 nanoparticles or Koumine@ZIF-8 nanoparticles is 50~200 mg / L.

[0052] In one embodiment, the spraying is performed during the seedling stage, root extension stage (rowing stage), or vigorous growth stage; preferably, during the seedling stage. Beneficial effects: The method for extracting Gelsemium alkaloid monomers of the present invention is simple, convenient, fast, and produces high-purity products. Furthermore, the obtained Gelsemium alkaloid monomers exhibit high biological activity when used as biopesticides, effectively preventing crop diseases and pests. Especially when prepared into nano-biopesticides, they show even higher activity, demonstrating broad application value in agriculture. Specific advantages are as follows: (1) The gelsemine A and gelsemine spores obtained in this invention showed good antibacterial effects against gray mold, and were superior to commercially available pesticides, such as the bio-based pesticide matrine (24.7%). Furthermore, the nano-gelsemine A (Gelsemine@ZIF-8) and nano-gelsemine spores (Koumine@ZIF-8) showed significantly better antibacterial effects against gray mold than gelsemine A (Gelsemine, 18.3%), gelsemine spores (Koumine, 19.1%), and the bio-based pesticide matrine (24.7%) at the same concentration. The nano-sized gelsemine alkaloids showed a more prominent antibacterial effect against gray mold. (2) The gelseminine A and gelseminine seeds obtained in this invention show good control effects on tobacco aphids and can effectively inhibit the growth of aphids; in particular, the aphid inhibition effect of the further formed nano gelseminine A (Gelsemine@ZIF-8) and nano gelseminine seeds (Koumine@ZIF-8) is significantly better than that of gelseminine A (Gelsemine, 20.3%), gelseminine seeds (Koumine, 17.9%) and biopesticide matrine (Matrine, 28.0%) at the same concentration, and the control effect on piercing-sucking aphids is better. Attached Figure Description

[0053] Figure 1 Gelsemium elegans A and Gelsemium elegans powder prepared in Example 1 of the present invention; (a) Gelsemium elegans A; (b) Gelsemium elegans; Figure 2 Chromatograms of the purity determination of gelseminine A and gelseminine prepared in Example 1 of the present invention by high performance liquid chromatography (HPLC); (a) gelseminine A; (b) gelseminine. Figure 3 The following are the 1H NMR purity detection spectra of gelseminine A and gelseminone prepared in Example 1 of this invention: (a) gelseminine A; (b) gelseminone. Figure 4 Transmission electron microscopy (TEM) images of Gelsemine@ZIF-8 and Koumine@ZIF-8 prepared in Example 2 of this invention; (a) Gelsemine@ZIF-8; (b) Koumine@ZIF-8; (c) Particle size distribution of Gelsemine@ZIF-8; (d) Particle size of Koumine@ZIF-8; Figure 5 Fourier transform infrared spectra of Gelsemine@ZIF-8 and Koumine@ZIF-8 prepared in Example 2 of this invention; Figure 6 SEM images of Gelsemine@ZIF-8 and Koumine@ZIF-8 prepared in Example 2 of this invention; (a) 500 nm image of ZIF-8-based nano-gelemine A; (b) 200 nm image of ZIF-8-based nano-gelemine A; (c) 100 nm image of ZIF-8-based nano-gelemine A; (d) 500 nm image of ZIF-8-based nano-gelemine; (e) 200 nm image of ZIF-8-based nano-gelemine; (f) 100 nm image of ZIF-8-based nano-gelemine. Figure 7 This is a graph showing the inhibitory effect of nano-Gelsemine@ZIF-8 and Koumine@ZIF-8 on the growth of gray mold spores after 120 hours of treatment in Example 3 of the present invention. Figure 8 This is a graph showing the control efficacy of nano-Gelsemine@ZIF-8 and Koumine@ZIF-8 against tobacco aphids in Example 3 of the present invention. Detailed Implementation

[0054] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the 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. The specific embodiments described below further illustrate the present invention.

[0055] Example 1 The extraction method of Gelsemium elegans alkaloids includes the following steps: (1) Cut the Gelsemium elegans into 2cm segments, and place the leaves and stems in a forced-air drying oven and dry them at 65℃ until constant weight; after drying, put the leaves and stems into a multi-functional pulverizer and pulverize them into powder samples. (2) Take 300 g of crushed Gelsemium elegans plant powder and place it in a beaker. Add 1500 mL of 3% sodium hydroxide solution to release the alkaloids. Stir well and let stand for 12 h. Add 3300 g of 75% ethanol at a material-to-liquid ratio of 1:11. Then place it in an ultrasonic oscillator for ultrasonic extraction for 1 h. After ultrasonic extraction, let it stand until there is obvious stratification in the beaker. Aspirate the supernatant and add 3300 g of 75% ethanol solution to the beaker. Then ultrasonically extract for 1 h. Repeat the above ultrasonic extraction steps once. Extract twice in total and combine the two extracts. (3) Use an 80 mm diameter Buchner funnel to filter and remove the precipitate in the extract of step (2) to obtain a clear extract. Place the extract in an eggplant-shaped flask and distill it under reduced pressure at 50°C to recover ethanol. Then, stop the rotary evaporation when the volume is about 1 / 10 of the initial volume or when there is no obvious ethanol odor to obtain a concentrated solution. (4) Adjust the pH of the concentrate to 2 with 3 mol / L hydrochloric acid solution, let it stand for 30 min, and then filter it with an 80 mm Buchner funnel. Collect the filtrate in a 500 mL vacuum filtration flask, extract the lipids in the filtrate with 50 mL dichloromethane, separate the organic phase and discard it; then adjust the pH of the filtrate to 11 with 25% sodium hydroxide solution, let it stand for 30 min, and then transfer the filtrate to a beaker. First, add 600 mL dichloromethane to the concentrate and sonicate for 30 min. After removing the concentrate, add another 500 mL dichloromethane and sonicate for 30 min. After removing the concentrate, add another 400 mL dichloromethane and sonicate for 30 min. Repeat this extraction process three times in total. Then combine the 1500 mL extracts. Combine the extracts in a 500 mL pear-shaped flask, remove the solvent under reduced pressure at 45 °C, and dry to obtain the crude extract. (5) Weigh 200 mg of the crude extract powder from step (4), prepare a 200 mg / mL crude extract sample solution with chromatographic grade acetonitrile, filter through a 0.45 μm organic filter membrane, and purify by preparative high performance liquid chromatography (HPLC) under the following conditions: column: YMC-PackProC18 (250 mm × 20 mm, 5 μm) column; column temperature: 30 ℃; detection wavelength: 254 nm; injection volume: 300 µL; mobile phase: phase A: 0.5% formic acid aqueous solution, phase B: acetonitrile, elution program: 0~4 min, 10%~10%B; 4~14 min, 10%~14%B; 14~25 min, 14%~24%B; 25~35 min, 24%~24%B; 35~40 min, 24%~90%B; 40~60 min, 90%~10%B; (6) The eluents of gelsemium oleracea and gelsemium oleracea were collected in sections. The product peaks of gelsemium oleracea and gelsemium oleracea were separated. The regions with higher purity were collected directly, and the mixed peaks containing the two products were prepared in a second process. The post-processing of the prepared products involved removing the solvent by vacuum distillation, drying the solvent by rotary evaporation, and then drying the samples in a forced-air drying oven to constant weight. Finally, gelsemium oleracea and gelsemium oleracea samples were successfully prepared.

[0056] Results Measurement 1. Sample purity testing Accurately weigh 10 mg of gelseminalis A standard (purity 99.21%) and gelseminalis seed standard (purity 99.48%) into a 10 mL volumetric flask, and dilute to the mark with acetonitrile (chromatographic grade) to prepare a 1 mg / mL standard solution. Pipette the 1 mg / mL gelseminalis A and gelseminalis seed standard solutions into 10 mL centrifuge tubes, and dilute with acetonitrile (chromatographic grade) to prepare solutions with concentrations of 0.5 μg / mL, 1 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, and 100 μg / mL, respectively, and perform liquid chromatography detection. Chromatographic conditions: column: YMC-PackProC18 (250 mm × 20 mm, 5 μm) column; column temperature: 30 ℃; detection wavelength: 254 nm. nm; Injection volume: 10µL; Mobile phase: Phase A: 0.5% formic acid aqueous solution, Phase B: acetonitrile, Elution program: 0–4 min, 10%–10% B; 4–14 min, 10%–14% B; 14–25 min, 14%–24% B; 25–35 min, 24%–24% B; 35–40 min, 24%–90% B; 40–60 min, 90%–10% B.

[0057] Weigh out 1 mg of gelsemin A and gelsemin sample prepared in Example 1 and place them in a 10 ml volumetric flask. Add acetonitrile (chromatographic grade) to the mark to prepare a 1 mg / ml sample solution. Transfer the sample solution into a 10 ml centrifuge tube and dilute it with acetonitrile (chromatographic grade) to a 100 μg / ml solution. Perform liquid chromatography detection using the same detection method as described above, and substitute the results into the standard curve to calculate the purity.

[0058] The results showed that the extraction rate of gelsemin A was 8.4% (gelsemin A / crude extract), the extraction rate of gelsemin seeds was 7.2% (gelsemin seeds / crude extract), the purity of gelsemin A was 97.1%, and the purity of gelsemin seeds was 98.4%.

[0059] 2. NMR identification of samples The gelsemin A and gelseminoid samples prepared in Example 1 were freeze-dried in 99.9% D₂O and deuterated, then tested using 99.95% D₂O as the solvent. Nuclear magnetic resonance (NMR) spectra were recorded using a Bruker Ascend 600 MHz spectrometer (Bruker, Germany) at 27 °C. The chemical shift of the proton NMR spectrum was referenced to the solvent residue peak (δH 4.79 ppm). NMR data were acquired and processed using Bruker software TopSpin 4.1.0. In the proton NMR experiment, the pulse program was zg30, the acquisition time was 2.75 s, the temperature was 27 °C, and the number of scans was 16; the results are as follows. Figure 3 As shown.

[0060] Example 2 The preparation of nano-gelemine (ZIF-8) includes the following: Dissolve 6.15 g of 2-methylimidazole in 45 ml of deionized water and sonicate for 10 min. Then add 50 mg of gelsemin A prepared in Example 1 to the 2-methylimidazole solution and stir magnetically at room temperature for 10 min. Next, dissolve 0.372 g of Zn(NO3)2·6H2O in 5 ml of deionized water and slowly add it dropwise to the 2-methylimidazole solution under magnetic stirring at 500 rpm (the molar ratio of Zn in the solution is...). 2+ The mixture was prepared by reacting 2-methylimidazole with water in a ratio of 1:60:2228 at room temperature for 30 min, then centrifuged at 10,000 rpm for 15 min, and then washed three times with deionized water and ethanol respectively to remove impurities from the ZIF-8 nanoparticles, thus obtaining Gelsemine@ZIF-8 nanoparticles.

[0061] Preparation of nano-Gelsemium koumine (Koumine@ZIF-8) includes the following: 6.15 g of 2-methylimidazole was dissolved in 45 ml of deionized water and sonicated for 10 min. Then, 50 mg of gelsemium elegans prepared in Example 1 was added to the 2-methylimidazole solution and magnetically stirred at room temperature for 10 min. Then, 0.372 g of Zn(NO3)2·6H2O was dissolved in 5 ml of deionized water and slowly added dropwise to the 2-methylimidazole solution under magnetic stirring at 500 rpm (the molar ratio of Zn in the solution was 1:1). 2+ The reaction mixture was prepared by reacting 2-methylimidazole with water in a ratio of 1:60:2228 at room temperature for 30 min, then centrifuged at 10,000 rpm for 15 min, and then washed three times with deionized water and ethanol respectively to remove impurities from the ZIF-8 nanoparticles, thus obtaining Koumine@ZIF-8 nanoparticles.

[0062] Structural identification and characterization The morphology of Gelsemine@ZIF-8 and Koumine@ZIF-8 prepared in Example 2 was analyzed by TEM. The results showed that both Gelsemine@ZIF-8 and Koumine@ZIF-8 nanoparticles exhibited a cubic crystal morphology, with average sizes of 263.66±16.37 nm and 277.31±22.98 nm, respectively. Figure 4 ).

[0063] Fourier transform infrared (FTIR) spectroscopy showed that ZIF-8 and both Gelsemine@ZIF-8 and Koumine@ZIF-8 nanomaterials shared the same characteristic peak (Zn). The N-stretching and C=N bending tests demonstrated that the structure of ZIF-8 remained unchanged after loading gelsemine A and gelsemine ions. Furthermore, an absorption peak of the C=C (1586 cm⁻¹) vibration of gelsemine A was observed in Gelsemine@ZIF-8, and absorption peaks caused by the C=C (1586 cm⁻¹) and COC (1078 cm⁻¹) bending vibrations of gelsemine ions were also observed in Koumine@ZIF-8. These characteristic peaks indicate that ZIF-8 nanoparticles successfully loaded gelsemine A and gelsemine ions. Figure 5 ).

[0064] Scanning electron microscope (SEM) images show ( Figure 6 The two ZIF-8-based nanogelseridin A and ZIF-8-based nanogelseridin atoms loaded with drugs have the same crystal structure as ZIF-8, exhibiting almost regular rhombic dodecahedral shapes. Overall, their crystal morphology is essentially the same as that of unloaded ZIF-8. This indicates that the self-assembly structure of the ZIF-8 crystals was not disrupted during drug loading.

[0065] Example 3 The inhibitory effects of nano-gelemine A (Gelsemine@ZIF-8) and nano-gelemine (Koumine@ZIF-8) on the growth of tomato gray mold (the pathogen of gray mold).

[0066] 10 mL of Gelsemine@ZIF-8 and Koumine@ZIF-8 at different concentrations (50, 100, 200 mg / L) were prepared, along with a pure water blank control and controls of 200 mg / L of gelsemine A, gelsemine spores, and the commercially available biopesticide matrine; each treatment was replicated in triplicate.

[0067] After sonication for 30 min, all treated samples were placed in a clean bench for UV sterilization for 30 min. The solution was then poured into partially solidified potato dextrose agar (PDA) medium, mixed thoroughly, and allowed to cool and solidify. Cylindrical hyphae (1 cm) were cut from the edge of the PDA mother medium using a punch and placed in each PDA medium. The medium was then placed in a 25°C biochemical incubator for growth. After 120 hours of treatment, the diameter of the hyphae was measured twice laterally at the bottom of the petri dish.

[0068] The results showed that, compared with the control group, 50 mg / L, 100 mg / L, and 200 mg / L nano-gelemine A (Gelsemine@ZIF-8) inhibited the diameter of gray mold plaques by 3.3%, 14.7%, and 37.0%, respectively, while 50 mg / L, 50 mg / L, and 200 mg / L nano-gelemine (Koumine@ZIF-8) inhibited the diameter of gray mold plaques by 2.9%, 16.5%, and 41.3%, respectively. The antibacterial effects of 200 mg / L nano-gelemine@ZIF-8 and Koumine@ZIF-8 were significantly better than those of gelemine A (18.3%), Koumine (19.1%), and the biopesticide matrine (24.7%) at the same concentrations. Figure 7 This indicates that nano-sized Gelsemium alkaloids significantly enhance the antibacterial effect against gray mold.

[0069] Example 4 Application of nano-gelemine@ZIF-8 and nano-gelemine@ZIF-8 in the control of tobacco aphids.

[0070] Tobacco seeds were sown in seedling trays, 15-20 seeds per cell, for a total of 50 cells. The trays were placed in a greenhouse (25°C, 60±5% relative humidity, 14 h / 10 h (day / night), 15000 lx light) for germination. After 15 days of germination, tobacco seedlings of similar growth were transplanted into flowerpots containing 0.5 kg of soil and placed randomly in the greenhouse.

[0071] Gelsemine@ZIF-8 and Koumine@ZIF-8 at concentrations of 50 mg / L, 100 mg / L, and 200 mg / L were selected, with a pure water blank control and traditional pesticide controls of 200 mg / L gelsemine A, gelsemine B, and the bio-based pesticide matrine. Each group was repeated three times. After the tobacco plants had grown for 30 days (15 days after transplanting), 10 mL of the above material solutions were sprayed evenly onto the surface of the tobacco leaves using a sprayer. After spraying, 20 adult tobacco aphids were placed on the leaves and wrapped with a 75 μm insect-proof net to prevent the aphids from escaping. The number of tobacco aphids was counted after 7 days of treatment.

[0072] The results showed that, compared with the control group, 50 mg / L, 100 mg / L, and 200 mg / L nano-gelemine@ZIF-8 reduced aphid populations by 29.2%, 38.4%, and 44.8%, respectively, while 50 mg / L, 100 mg / L, and 200 mg / L nano-gelemine@ZIF-8 reduced aphid populations by 28.8%, 42.7%, and 54.7%, respectively. The aphid-inhibiting effects of 200 mg / L nano-gelemine@ZIF-8 and Koumine@ZIF-8 were significantly better than those of the same concentrations of gelemine (20.3%), Koumine (17.9%), and the biopesticide matrine (28.0%). Figure 8 This indicates that nano-sized Gelsemium alkaloids have a better control effect on piercing-sucking aphids.

[0073] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for extracting monomers of Gelsemium elegans alkaloids, characterized in that, The method includes the following steps: (1) Add sodium hydroxide solution to the Gelsemium sample, stir well and let stand, then add ethanol solution, extract by ultrasonication, and collect the supernatant; repeat 2-3 times, combine the supernatants, and the extract is the extract; (2) The extract from step (1) is concentrated by rotary evaporation, and the pH of the concentrate is adjusted to 2-3. After standing, it is filtered. The filtrate is extracted with dichloromethane to remove the organic phase. Then the pH of the filtrate is adjusted to 10-11 and allowed to stand. Then it is extracted with dichloromethane again, the extract is collected, the solvent is recovered under reduced pressure, and it is dried to obtain the crude extract. (3) Dissolve the crude extract powder obtained in step (3) in acetonitrile and filter it through an organic filter membrane. The filtrate is separated and purified by preparative high performance liquid chromatography to obtain gelsemin A monomer and gelsemin A monomer respectively.

2. The method according to claim 1, characterized in that, The sodium hydroxide solution in step (1) has a mass fraction of 3-5%.

3. The method according to claim 1, characterized in that, In step (1), the ratio of the gelsevieria sample to the sodium hydroxide solution is 1~3:10~15g:mL.

4. The method according to claim 1, characterized in that, The amount of ethanol solution added in step (1) is 10 to 15 times the mass of the Gelsemium elegans sample.

5. The method according to claim 1, characterized in that, The detection conditions for the preparative high performance liquid chromatograph in step (3) are as follows: chromatographic column: YMC-PackProC18 250 mm × 20 mm, 5 μm; Column temperature: 30 ℃; Detection wavelength: 254 nm; Injection volume: 300 µL; Mobile phase: Phase A: 0.5% formic acid aqueous solution, Phase B: acetonitrile, Elution program: 0–4 min, 10%–10% B; 4–14 min, 10%–14% B; 14–25 min, 14%–24% B; 25–35 min, 24%–24% B; 35–40 min, 24%–90% B; 40–60 min, 90%–10% B.

6. The gelseminine alkaloid monomers extracted by the method according to any one of claims 1 to 5.

7. The gelseminine alkaloid monomer according to claim 6, characterized in that, It includes gelsemin A monomer and gelsemin A monomer; the structures of the gelsemin A monomer and gelsemin A monomer are shown in Formulas I and II: Formula I; Formula II.

8. The application of the gelseminine alkaloid monomer as described in claim 6 or 7 in the prevention and control of crop diseases and pests.

9. A method for preparing gelsemium alkaloid nanoparticles, characterized in that, The method includes the following: Dissolve 2-methylimidazole in water and add the gelsemium alkaloid monomer as described in claim 6 or 7. Stir magnetically at room temperature to obtain a 2-methylimidazole solution. Then dissolve Zn(NO3)2·6H2O in water and slowly add it dropwise to the 2-methylimidazole solution under stirring. React at room temperature. After the reaction is complete, centrifuge and wash the precipitate to obtain the final product.

10. The method according to claim 9, characterized in that, The mass ratio of the 2-methylimidazole, gelsemium alkaloid monomer, and water is 6~8:0.05:40~50.

11. The method according to claim 10, characterized in that, The mass ratio of Zn(NO3)2·6H2O to 2-methylimidazole is 0.1~0.5:2~8.

12. The method according to claim 10, characterized in that, The reaction time is 30-60 minutes.

13. The gelsemium alkaloid nanoparticles prepared by the method according to any one of claims 9 to 12, characterized in that, The gelsemine alkaloid nanoparticles are either gelsemine@ZIF-8 nanoparticles or koumine@ZIF-8 nanoparticles.

14. The gelsemium alkaloid nanoparticles according to claim 13, characterized in that, The average size of the Gelsemine@ZIF-8 nanoparticles is 240~280nm; the average size of the Koumine@ZIF-8 nanoparticles is 250~310nm.

15. The application of the gelsemium alkaloid nanoparticles according to claim 13 or 14 in the prevention and control of crop diseases and pests.

16. A botrytis cinerea inhibitor, characterized in that, The botrytis cinerea inhibitor contains active ingredients of gelseminine A and / or gelseminine.

17. The botrytis cinerea inhibitor according to claim 16, characterized in that, The gelseminine A comprises gelseminine A monomers or gelseminine A nanoparticles; the gelseminone comprises gelseminone monomers or gelseminone nanoparticles.

18. The application of a gelsemium alkaloid in the control of tobacco aphids, characterized in that, The application involves preparing a solution of gelsemium alkaloids and spraying it onto tobacco leaves.

19. The application according to claim 18, characterized in that, The gelseminoids include any one or more of gelseminine A monomer, gelsemine@ZIF-8 nanoparticles, gelseminine A monomer, and Koumine@ZIF-8 nanoparticles.

20. A method for improving the control effect of tobacco aphids, characterized in that, The method involves preparing the gelsemium alkaloid nanoparticles according to claim 13 or 14 into a solution and spraying it onto tobacco leaves.

Images