A diterpenoid alkaloid compound and application thereof in preventing and treating radix codonopsis root rot

Abstract

This invention relates to the technical field of traditional Chinese medicine compounds, specifically a diterpenoid alkaloid compound for preventing and treating root rot in Codonopsis pilosula. The diterpenoid alkaloid compound is neoaconitine, and its structural formula is shown in Figure 1. The neoaconitine is a white amorphous powder. 1 H-NMR signals included three groups of hydrogen atoms on the aromatic ring, one monosubstituted acetyl group, four methoxy hydrogen atoms, and one group of N-methyl hydrogen atoms. 13 C-NMR has a specific chemical shift value. This invention has a strong inhibitory effect on Fusarium oxysporum, the pathogen causing root rot in Codonopsis pilosula, and can effectively inhibit the growth and reproduction of pathogenic mycelia, blocking the occurrence and spread of the disease from the source, significantly reducing the incidence and severity of root rot in Codonopsis pilosula, ensuring the normal growth of Codonopsis pilosula, and improving yield and quality.

Description

Technical Field

[0001] This invention relates to the technical field of traditional Chinese medicine compounds, specifically to a diterpenoid alkaloid compound and its application in the prevention and treatment of root rot in Codonopsis pilosula. Background Technology

[0002] Codonopsis pilosula, a traditional and precious Chinese medicinal herb, holds an important position in medicinal and health-care fields. Its artificial cultivation scale has been expanding year by year, becoming a significant pillar of the agricultural economy in some regions. However, root rot is an increasingly prominent problem in Codonopsis pilosula cultivation, severely hindering the healthy development of the industry. This disease is mainly caused by Fusarium oxysporum, initially manifesting as localized root rot, which then gradually spreads to the entire root system, leading to weakened plant growth, wilting, and death, ultimately resulting in significant yield reduction or even total crop failure. It also affects the content and quality of medicinal components in Codonopsis pilosula, causing huge economic losses to growers.

[0003] Currently, the control methods for Codonopsis pilosula root rot are relatively limited, mainly relying on chemical pesticide spraying or soil disinfection. However, long-term use of chemical pesticides easily leads to drug resistance in pathogens, causing the control effect to decline year by year, and also causing environmental problems such as soil pollution and pesticide residues, which is inconsistent with the concept of green agriculture development. Although biological control methods have the advantage of being environmentally friendly, the antibacterial effects of existing strains or biological agents are unstable and greatly affected by environmental factors, making it difficult to meet the control needs of large-scale planting. In addition, although some Chinese herbal extracts have been tried for disease control, most of them have problems such as unclear active ingredients, poor antibacterial effects, and unclear mechanisms of action, and have failed to achieve industrial application. Therefore, developing a highly efficient, safe, and clearly defined agent for the control of Codonopsis pilosula root rot has become a key issue that urgently needs to be addressed in the current industrial development. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by proposing a diterpenoid alkaloid compound and its application in the prevention and treatment of root rot in Codonopsis pilosula.

[0005] A diterpenoid alkaloid compound for the prevention and treatment of root rot in Codonopsis pilosula, wherein the diterpenoid alkaloid compound is neoaconitine, and the structural formula of neoaconitine is shown in the attached figure. Figure 1 As shown; the neoaconitine is a white amorphous powder, its 1 H-NMR signals included three groups of hydrogen atoms on the aromatic ring, one monosubstituted acetyl group, four methoxy hydrogen atoms, and one group of N-methyl hydrogen atoms. 13 C-NMR has specific chemical shift values.

[0006] According to the application of the diterpenoid alkaloid compound neoaconitine in the preparation of a medicine for preventing and treating root rot of Codonopsis pilosula, the root rot of Codonopsis pilosula is caused by Fusarium oxysporum; the inhibitory activity of neoaconitine against Fusarium oxysporum is manifested by destroying the cell wall of Fusarium oxysporum, inhibiting the activity of β-1,3-glucanase and chitinase in Fusarium oxysporum, or inhibiting the activity of malate dehydrogenase and succinate dehydrogenase in Fusarium oxysporum.

[0007] Preferably, the pharmaceutical preparation comprises neoaconitine and a pharmaceutically acceptable excipient selected from at least one of fillers, dispersants, wetting agents, binders, or solvents; the filler is kaolin or diatomaceous earth, the dispersant is sodium lignosulfonate or naphthalenesulfonate formaldehyde condensate, and the wetting agent is sodium dodecylbenzenesulfonate or fatty alcohol polyoxyethylene ether.

[0008] Preferably, the agent contains 0.01% to 5% by mass of neoaconitine; wherein the agent used for root irrigation contains 0.1% to 5% by mass of neoaconitine, and the agent used for foliar spraying contains 0.01% to 0.5% by mass of neoaconitine.

[0009] Preferably, the dosage form of the agent is a wettable powder, a suspension, or an emulsifiable concentrate; the wettable powder contains 5% to 10% by mass of dispersant and 2% to 5% by mass of wetting agent; the suspension contains 3% to 8% by mass of dispersant and 0.1% to 0.5% by mass of thickener; and the emulsifiable concentrate contains 20% to 50% by mass of solvent and 5% to 15% by mass of emulsifier.

[0010] Preferably, the agent containing the diterpenoid alkaloid compound of claim 1, namely neoaconitine, is applied to the Codonopsis pilosula plant or its growth environment, wherein the growth environment includes cultivation soil or cultivation substrate; after the agent is applied, it can reduce the rot rate of Codonopsis pilosula roots or increase the survival rate of Codonopsis pilosula plants.

[0011] Preferably, the method of application is root irrigation, foliar spraying, or soil drenching; when irrigating the roots, the agent is applied to the soil within a range of 10cm to 15cm around the roots of Codonopsis pilosula, with 50mL to 200mL of agent applied to each Codonopsis pilosula plant; when foliar spraying, the agent is evenly sprayed on both sides of the Codonopsis pilosula leaves until uniform droplets are formed on the leaf surface and do not drip.

[0012] Preferably, when the agent is applied, the concentration of neoaconitine in the agent is 0.01 mg / mL to 0.1 mg / mL; the agent is applied once every 7 to 14 days, for 2 to 3 consecutive times; when Codonopsis pilosula is in the seedling stage, the application concentration is 0.01 mg / mL to 0.05 mg / mL, and when it is in the mature stage, the concentration is 0.05 mg / mL to 0.1 mg / mL.

[0013] A method for preparing the pharmaceutical agent includes the following steps: (1) Extraction: The dried Aconitum carmichaelii was pulverized to a particle size of 20 to 40 mesh, and extracted three times by soaking in industrial methanol. The amount of methanol used each time was 5 to 8 times the mass of Aconitum carmichaelii, and the soaking time was 7 days each time. The three extracts were combined and concentrated under reduced pressure at a temperature of 40℃ to 50℃ and a vacuum degree of -0.08MPa to -0.06MPa to obtain the methanol extract. (2) Preliminary separation: The methanol extract was dissolved in hot water at 60°C, and the amount of hot water was 10 to 15 times the mass of the methanol extract. The crude extract was performed three times with petroleum ether, and the amount of petroleum ether was the same as the amount of hot water. The aqueous phase after each extraction was collected and combined to obtain the total aqueous phase. (3) Acid extraction: Add 2% hydrochloric acid solution to the total aqueous phase, adjust the pH value to 1 to 3, and extract with dichloromethane three times. The amount of dichloromethane used is the same as the amount of total aqueous phase. Combine the three dichloromethane extracts and concentrate under reduced pressure at a temperature of 35℃ to 45℃ and a vacuum degree of -0.08MPa to -0.06MPa to obtain dichloromethane acid extract; (4) Column chromatography purification: The dichloromethane acid extract was dissolved in a 50% ethanol solution and loaded onto a macroporous resin column of type D101 or AB-8. It was eluted sequentially with 30% ethanol, 50% ethanol, 70% ethanol and 90% ethanol solutions. The amount of ethanol used for each concentration was 3 to 5 times the column volume. Four components were collected. The activity was tracked by the mycelial growth rate method to determine the active component containing neoaconitine. The active component was concentrated and loaded onto a normal phase silica gel column with a particle size of 100 to 200 mesh. It was eluted with a mixture of dichloromethane and methanol with a volume ratio gradually adjusted from 200:1 to 30:1. The elution flow rate was 1 mL / min to 2 mL / min. (5) Crystallization and formulation: The composition of the fractions obtained by normal-phase silica gel column chromatography is determined by thin-layer chromatography. The fractions containing neoaconitine are combined and concentrated to 1 / 5 to 1 / 3 of the original volume at a temperature of 30°C to 40°C. The fractions are then placed in a refrigerator at 4°C for 24 to 48 hours to precipitate white amorphous crystals, which are the diterpenoid alkaloids described in claim 1. The crystals are mixed with pharmaceutically acceptable excipients and the formulation is prepared according to conventional formulation processes.

[0014] Preferably, the particle size of the dried aconite in step (1) is 20 to 30 mesh; in step (4), the volume ratio of dichloromethane to methanol is adjusted every 5 column volumes, gradually decreasing from 200:1 to 30:1.

[0015] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention has a strong inhibitory effect on Fusarium oxysporum, the pathogen causing root rot of Codonopsis pilosula. It can effectively inhibit the growth and reproduction of pathogenic mycelium, block the occurrence and spread of the disease from the source, significantly reduce the incidence and severity of root rot of Codonopsis pilosula, ensure the normal growth of Codonopsis pilosula, and improve yield and quality.

[0016] 2. This invention exerts its antibacterial effect through multiple pathways. It can destroy the cell wall structure of pathogens, causing abnormal hyphal morphology and growth inhibition, inhibit the activity of cell wall synthesis-related enzymes, and affect the antioxidant system and energy metabolism process in pathogens, thus disrupting their physiological metabolic balance. This multi-target mechanism makes it difficult for pathogens to develop drug resistance through gene mutation, thereby extending the lifespan of the drug.

[0017] 3. This invention is derived from the traditional Chinese medicine Aconitum carmichaelii, which is obtained through a reasonable extraction and separation process. It is a natural active ingredient. Compared with chemical pesticides, it has no significant adverse effects on the soil environment, surrounding ecosystem, and non-target organisms. Moreover, the residue in Codonopsis pilosula plants is extremely low, which meets the requirements of green Chinese medicinal herb cultivation and ensures the quality and safety of the product.

[0018] 4. The compound extraction and separation process of this invention is scientific and reasonable, simple to operate, and can achieve efficient enrichment and purification of target components. Furthermore, the compound structure is clearly defined, facilitating subsequent large-scale production and quality control. This compound can be further prepared into suitable pesticide formulations for field control in Codonopsis pilosula cultivation. It also provides new ideas and technical support for the control of Fusarium diseases in other crops, possessing significant economic and social value. Attached Figure Description

[0019] Figure 1 Here is the structural diagram of the compound neoaconitine; Figure 2 This is a flowchart of the compound separation process; Figure 3 For the compound neoaconitine 1 H-NMR spectrum; Figure 4 For the compound neoaconitine 13 C-NMR spectrum; Figure 5 The graph shows the antibacterial activity of the compound neoaconitine against the pathogen causing root rot of Codonopsis pilosula. Figure 6 Electron micrographs of normal Fusarium oxysporum hyphae and hyphae treated with the chemical. Figure 7 This is a graph showing the determination of the relevant enzyme activity. Detailed Implementation

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0021] Example 1: Extraction and structural identification of the target alkaloid compound (1) Extraction and separation: Eight kilograms of dried Aconitum carmichaelii were first crushed, then soaked and extracted three times with industrial methanol for seven days each time, followed by vacuum concentration to obtain the methanol extract.

[0022] The methanol extract was dissolved in hot water at 60°C and crudely extracted three times with petroleum ether to obtain petroleum ether extract M and the remaining aqueous phase.

[0023] The aqueous phase was adjusted to pH 1-3 with 2% hydrochloric acid solution. The mixture was extracted three times with dichloromethane and concentrated under reduced pressure to obtain dichloromethane acid extract N, with a mass of 25.96 g. Dichloromethane acid extract N was then separated and eluted by macroporous resin column chromatography to obtain four fractions of extract Fr.(AD).

[0024] The different fractions Fr.(AD) were obtained, and the target active compound was identified in fraction Fr.A (7.77 g) by activity tracking combined with normal phase silica gel column chromatography, reverse phase silica gel column chromatography, and dextran gel chromatography.

[0025] Normal-phase silica gel column chromatography was performed on the fraction Fr.A containing the target active compound. Based on the thin-layer chromatography method, dichloromethane:methanol (200:1-30:1) was used as the eluent. The fractions were collected, spotted onto a plate, combined and concentrated to obtain 4 fractions. The first fraction Fr.A1 precipitated crystals, which yielded the diterpenoid alkaloid compound.

[0026] (2) Structural identification of compounds The structural formula of the compound is as follows: Figure 1 As shown, 1 H-NMR spectrum and 13 The C-NMR spectra are as follows: Figure 3-4 As shown. Analysis revealed that the compound is a white amorphous powder. 1 H-NMR (500MHz, CDCl3) showed: The hydrogen signals on three groups of aromatic rings are δH 7.99 (2H, d, J = 7.8 Hz), 7.54 (1H, t, J = 7.1 Hz), and 7.42 (2H, t, J = 7.9 Hz); one monosubstituted acetyl group has a δH 1.33 (3H, s, OCH3); four methoxy groups have hydrogen signals δH 3.12, 3.23, 3.25, and 3.69 (each 3H, s); and one group of nitrogen-methyl groups has a hydrogen signal of 2.29 (m, 3H, N-CH3).

[0027] 13 C-NMR(125MHz, CDCl3)c:172.5,166.1,133.4,129.8,129.6,128.7,91.9,90.0,83.2,82.4,78.9,76.1,7 4.1,71.0,62.2,61.1,59.1,58.0,56.4,49.9,49.4,46.5,44.3,43.7,43.5,42.5,40.8,35.8,34.2,21.5.

[0028] The above data is consistent with the previously reported data on neoaconitine, thus confirming that they are the same compound.

[0029] Example 2: Antibacterial Experiment of Chemical Components of Aconitum carmichaelii (1) The mycelial growth rate method was used to determine the antibacterial activity of Aconitum carmichaelii dichloromethane extract against the pathogen causing root rot of Codonopsis pilosula. The specific operation was as follows: 10 mg of the monomer compound was weighed and added to 0.2 mL of dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 50 mg / mL. Then, 0.055 mL of the stock solution was added to 50 mL of potato dextrose agar (PDA) medium and mixed thoroughly to prepare a drug-containing medium with a test concentration of 50 μg / mL. In the PDA medium of the control group, an equal amount of DMSO as in the stock solution was added. After that, the medium of each group was poured into petri dishes and allowed to cool and solidify. Using a 5 mm diameter punch, mycelial cakes were taken from the colonies of the tested pathogen and transferred to the center of the medium. Each treatment group was replicated three times. The petri dishes were incubated at 25 °C until the colonies of the control group just covered the entire medium. At this point, the growth diameter of the tested colonies is measured using the cross-cross method, and the inhibition rate is calculated.

[0030] (2) Formula for calculating inhibition rate Mycelial growth (cm) = Mycelial growth measurement diameter - Mycelial cake diameter

[0031] The antibacterial results of the chemical components of Aconitum carmichaelii are shown in Table 1 and... Figure 5 As shown.

[0032] Neoaconitine virulence regression equation and EC50 against Fusarium oxysporum DS-QNN2 Table 1:

[0033] As shown in Table 1, neoaconitine has strong antibacterial activity against Fusarium oxysporum. Example 3: Antibacterial experiment of neoaconitine against root rot of Codonopsis pilosula (1) Based on the EC50 value of neoaconitine against DS-QNN2 Fusarium oxysporum, dimethyl sulfoxide (DMSO) was dissolved in the solution and drug-containing culture medium was prepared in a clean bench according to the EC50 concentration gradient before the PDA medium condensed. The activated mycelial cake of the fungus was obtained using a 5.0 mm sterile punch and inoculated into the center of a 90 mm culture dish with the mycelial side down. A drug-free PDA plate was set as a blank control. The culture dishes were sealed and inverted in a constant temperature incubator at 25 ℃ for 5 days. The culture was terminated when the mycelium of the control group filled the plate. The mycelium of each group was scraped and placed in a 10 mL sterile centrifuge tube for testing. Take appropriate amounts of mycelia from the treatment group and the control group, wash three times with PBS solution (500mL containing 4g sodium chloride, 0.1g potassium chloride, 0.71g sodium dihydrogen phosphate, and 0.12g potassium dihydrogen phosphate), and aspirate the remaining liquid. Immediately transfer to fixative: first fix with 2.5%-5% glutaraldehyde at 4℃ for several hours to overnight, rinse with buffer, and then fix with 1% osmium tetroxide for 1-2 hours. After fixation, dehydrate with a gradient of 30%→50%→70%→80%→90%→95%→100% alcohol, holding at each concentration for 15-30 minutes; after freeze-drying to remove organic solvents, use an ultramicrotome to cut the embedded samples into 50-100nm ultrathin sections and fix them on the electron microscope stage. First, perform a 2000× low-magnification panoramic scan, select typical regions for marking, and then observe subcellular structures at 5000× high magnification.

[0034] The effects of the compound neoaconitine on bacterial cell structure are as follows: Figure 6 As shown, The results showed that the hyphae of normal *Fusarium oxysporum* were relatively regular in morphology and of relatively uniform thickness. The hyphae treated with the agent underwent significant changes, with numerous irregular protrusions on the surface, and some hyphae exhibiting constriction and twisting. This indicates that the agent disrupted the normal growth morphology of the hyphae, hindering their normal growth and development. The cell wall of *Fusarium oxysporum* plays a crucial role in maintaining hyphal morphology. The agent inhibited the activity of enzymes or metabolic pathways related to cell wall synthesis, preventing the normal synthesis of new cell wall substances. This affected the strength and uniformity of the cell wall, potentially leading to protrusions and wrinkling.

[0035] (2) Determination of related enzyme activities Using the EC50 of neoaconitine against *Fusarium oxysporum* DS-QNN2, a pathogen causing root rot in *Codonopsis pilosula*, as a baseline, solutions with concentrations of 20 μg / mL, 40 μg / mL, and 80 μg / mL were prepared. After preparation, 2 mL of each solution was added to a 50 mL Erlenmeyer flask containing 18 mL of LPDA liquid medium. *Fusarium oxysporum* mycelial cakes of *DS-QNN2* were pre-prepared using a 5 mm perforator. Five mycelial cakes were added to each flask containing the prepared medium. 1 mL of sterile distilled water was used as a control. Each treatment was repeated three times. After standing for 12 hours, the cultures were incubated at 25°C and 150 rpm for 3 days, followed by sampling. During sampling, the cultures were filtered through sterile silk cloth to obtain mycelia and filtrate. Next, the obtained filtrate was centrifuged at 10,000 rpm for 10 minutes at 4°C, and then stored at -20°C. The *Fusarium oxysporum* hyphae obtained after filtration were blotted dry with filter paper and then placed in a sterile mortar. Enzyme extraction solution was added to the mortar, and the hyphae were ground into a homogenate under ice bath conditions. The supernatant was then aspirated to obtain the crude enzyme solution. This solution was used for subsequent determination of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and malondialdehyde (MDA) content. These four enzymes can be extracted using the same extraction solution. The remaining four enzyme activities—maltate dehydrogenase (MDH), succinate dehydrogenase (SDH), β-1,3-glucanase, and chitinase—were extracted using the extraction solutions and experimental procedures specified in their respective kits.

[0036] The results of the relevant enzyme activity assay are shown in the figure below. Figure 7 As shown Experimental results show that the antibacterial mechanism of DS-QNN2 is through inhibiting the activity of β-1,3-glucanase and chitinase, thereby disrupting the fungal cell wall, causing hydrolytic imbalance, severe membrane lipidization, increased MDA content, and cell membrane damage; the antioxidant system is stressed, resulting in excessive accumulation of hydrogen peroxide in the fungus, decreased CAT enzyme activity, and reduced antioxidant capacity; during the TAC cycle, the activities of MDH and SDH enzymes are inhibited, ATP synthesis is reduced, and hyphal growth is slowed.

[0037] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. A diterpenoid alkaloid compound for preventing and treating root rot in Codonopsis pilosula, characterized in that, The diterpenoid alkaloid compound is neoaconitine, and the structural formula of neoaconitine is shown in Figure 1; neoaconitine is a white amorphous powder. 1 H-NMR signals included three groups of hydrogen atoms on the aromatic ring, one monosubstituted acetyl group, four methoxy hydrogen atoms, and one group of N-methyl hydrogen atoms. 13 C-NMR has specific chemical shift values.

2. The application of the diterpenoid alkaloid compound of claim 1 in the preparation of an agent for preventing and treating root rot of Codonopsis pilosula, wherein the root rot of Codonopsis pilosula is caused by Fusarium oxysporum; the inhibitory activity of the neoaconitine against Fusarium oxysporum is manifested by destroying the cell wall of Fusarium oxysporum, inhibiting the activity of β-1,3-glucanase and chitinase in Fusarium oxysporum, or inhibiting the activity of malate dehydrogenase and succinate dehydrogenase in Fusarium oxysporum.

3. An application according to claim 2, characterized in that, The pharmaceutical preparation comprises neoaconitine and pharmaceutically acceptable excipients selected from at least one of fillers, dispersants, wetting agents, binders, or solvents; the filler is kaolin or diatomaceous earth, the dispersant is sodium lignosulfonate or naphthalenesulfonate formaldehyde condensate, and the wetting agent is sodium dodecylbenzenesulfonate or fatty alcohol polyoxyethylene ether.

4. An application as described in claim 3, characterized in that, The agent contains 0.01% to 5% by mass of neoaconitine; the agent used for root irrigation contains 0.1% to 5% by mass of neoaconitine, and the agent used for foliar spraying contains 0.01% to 0.5% by mass of neoaconitine.

5. An application as described in claim 3, characterized in that, The dosage form of the agent is a wettable powder, a suspension, or an emulsifiable concentrate; the wettable powder contains 5% to 10% by mass of dispersant and 2% to 5% by mass of wetting agent; the suspension contains 3% to 8% by mass of dispersant and 0.1% to 0.5% by mass of thickener; the emulsifiable concentrate contains 20% to 50% by mass of solvent and 5% to 15% by mass of emulsifier.

6. A method for preventing and controlling root rot in Codonopsis pilosula, characterized in that, The agent containing the diterpenoid alkaloid compound of claim 1, namely neoaconitine, is applied to the Codonopsis pilosula plant or its growth environment, wherein the growth environment includes cultivation soil or cultivation substrate; after the agent is applied, the rate of root rot of Codonopsis pilosula or the survival rate of Codonopsis pilosula plant can be reduced or increased.

7. The method of claim 6, characterized in that, The application methods are root irrigation, foliar spraying, or soil application. When irrigating the roots, apply the agent to the soil within a 10cm to 15cm radius around the roots of the Codonopsis pilosula, using 50mL to 200mL of the agent per plant. When foliar spraying, spray the agent evenly on both sides of the Codonopsis pilosula leaves until uniform droplets are formed on the leaf surface and do not drip.

8. The method of claim 6, characterized in that, When the agent is applied, the concentration of neoaconitine in the agent is 0.01 mg / mL to 0.1 mg / mL; the agent is applied once every 7 to 14 days, for 2 to 3 consecutive times; when Codonopsis pilosula is in the seedling stage, the application concentration is 0.01 mg / mL to 0.05 mg / mL, and when it is in the mature stage, the concentration is 0.05 mg / mL to 0.1 mg / mL.

9. A method for preparing the pharmaceutical agent according to claim 2, characterized in that, Includes the following steps: (1) Extraction: The dried Aconitum carmichaelii was pulverized to a particle size of 20 to 40 mesh, and extracted three times by soaking in industrial methanol. The amount of methanol used each time was 5 to 8 times the mass of Aconitum carmichaelii, and the soaking time was 7 days each time. The three extracts were combined and concentrated under reduced pressure at a temperature of 40℃ to 50℃ and a vacuum degree of -0.08MPa to -0.06MPa to obtain the methanol extract. (2) Preliminary separation: The methanol extract was dissolved in hot water at 60°C, and the amount of hot water was 10 to 15 times the mass of the methanol extract. The crude extract was performed three times with petroleum ether, and the amount of petroleum ether was the same as the amount of hot water. The aqueous phase after each extraction was collected and combined to obtain the total aqueous phase. (3) Acid extraction: Add 2% hydrochloric acid solution to the total aqueous phase, adjust the pH value to 1 to 3, and extract with dichloromethane three times. The amount of dichloromethane used is the same as the amount of total aqueous phase. Combine the three dichloromethane extracts and concentrate under reduced pressure at a temperature of 35℃ to 45℃ and a vacuum degree of -0.08MPa to -0.06MPa to obtain dichloromethane acid extract; (4) Column chromatography purification: The dichloromethane acid extract was dissolved in a 50% ethanol solution and loaded onto a macroporous resin column of type D101 or AB-8. It was eluted sequentially with 30% ethanol, 50% ethanol, 70% ethanol and 90% ethanol solutions. The amount of ethanol used for each concentration was 3 to 5 times the column volume. Four components were collected. The activity was tracked by the mycelial growth rate method to determine the active component containing neoaconitine. The active component was concentrated and loaded onto a normal phase silica gel column with a particle size of 100 to 200 mesh. It was eluted with a mixture of dichloromethane and methanol with a volume ratio gradually adjusted from 200:1 to 30:

1. The elution flow rate was 1 mL / min to 2 mL / min. (5) Crystallization and formulation: The composition of the fractions obtained by normal-phase silica gel column chromatography is determined by thin-layer chromatography. The fractions containing neoaconitine are combined and concentrated to 1 / 5 to 1 / 3 of the original volume at a temperature of 30°C to 40°C. The fractions are then placed in a refrigerator at 4°C for 24 to 48 hours to precipitate white amorphous crystals, which are the diterpenoid alkaloids described in claim 1. The crystals are mixed with pharmaceutically acceptable excipients and the formulation is prepared according to conventional formulation processes.

10. The method of claim 9, characterized in that, In step (1), the particle size of the dried aconite is 20 to 30 mesh; in step (4), during the elution process, the volume ratio of dichloromethane to methanol is adjusted every 5 column volumes, gradually decreasing from 200:1 to 30:1.

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