An alkaloid compound, composition, preparation method and application

Abstract

The application relates to an alkaloid compound, a composition, a preparation method and application, and relates to the technical field of medicines.The alkaloid compound comprises a compound as shown in formula (I) or a medicinal salt or a medicinal ester derivative of formula (I): The new alkaloid compound provided by the application is verified to have enzyme inhibition activity, wherein the IC50 value is 1.4758+ / -0.013 mg / mL; has insecticidal activity, when the concentration is 1 mg / mL of an aqueous solution, the killing effect on citrus psylla is 48.70+ / -0.36%; and has antibacterial activity, wherein the minimum inhibitory concentration (MIC) of staphylococcus aureus is 512 mu g / mL, and the minimum inhibitory concentration (MIC) of hemolytic streptococcus B is 1024 mu g / mL.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to an alkaloid compound, composition, preparation method, and application. Background Technology

[0002] Tyrosinase is widely distributed in organisms and is a key rate-limiting enzyme catalyzing melanin synthesis. It catalyzes the conversion of L-tyrosine (L-Tyr) to L-DOPA, and then oxidizes L-DOPA to form dopaquinone. In these reactions, the enzyme is referred to as tyrosinase monophenolase and tyrosinase diphenolase, respectively. Tyrosinase inhibitors are important ingredients in skin whitening products, currently mainly including kojic acid and hydroquinone, which have certain side effects on the human body. Natural polyphenols found in plants can safely and effectively inhibit tyrosinase activity, making them a focus of skin whitening product research and development in recent years.

[0003] Humans have long used plants such as tobacco, stemona, veratrum, sophora flavescens, and tripterygium wilfordii to kill insects. Alkaloid insecticides are a class of plant-based insecticides that have been widely used and studied. Currently, more than 6,000 alkaloids have been isolated from plants. Alkaloids are important lead compounds for the synthesis of new insecticides. Using nicotine as a template, a new class of highly effective, low-toxicity, and uniquely mechanism-of-action nicotine insecticides has been developed. The citrus psyllid (Diaphori nacitri Kuwayama), belonging to the order Homoptera and family Chermidae, is a major pest of new shoots of citrus, lemon, wampee, Murraya paniculata, and wolfberry, and is also the only natural vector for the transmission of citrus Huanglongbing (HLB). Control of citrus psyllids mainly relies on alkaloid insecticides such as veratrum alkaloid preparations, matrine emulsions, and nicotine microcapsule suspensions. However, the effective utilization of plant-derived natural insecticides in actual production remains limited. Therefore, developing more plant-derived alkaloid insecticides for citrus psyllids is particularly important for the effective control of citrus psyllids and citrus Huanglongbing (HLB).

[0004] In recent years, due to the overuse of antibiotics, pathogenic microorganisms have continuously evolved and mutated to adapt to environmental changes, leading to an increasing resistance to traditional antibiotics and the emergence of multidrug-resistant bacteria. The infectious diseases they cause seriously threaten human health, posing a significant challenge to the efficacy and speed of antibiotic development. Therefore, there is an urgent need to find effective antimicrobial drugs. Natural products have always been an important source of new drug development, possessing characteristics such as multiple targets and low resistance rates. Perhaps finding lead compounds with antimicrobial activity from natural products can open up new avenues for antibiotic development.

[0005] Studies have shown that the endophytic fungus in the tuberous roots of *Stephania kwangsiensis* possesses certain antibacterial activity. *Stephania kwangsiensis*, belonging to the genus *Stephania* in the family Menispermaceae, is a perennial herbaceous deciduous vine that can grow to over 3 meters in length. Its tuberous roots are typically flattened-globose. The leaves are alternate, papery, with petioles peltate at the base of the leaf (1–1.5 cm), and are 4–9 cm long. The leaf blades are triangular-orbicular to nearly orbicular. It is mainly produced in northwestern and southwestern Guangxi, my country, growing in limestone mountain thickets. Currently, reports on the chemical composition of *Stephania kwangsiensis* are incomplete; therefore, this invention provides an alkaloid compound, a composition, a preparation method, and its applications. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide an alkaloid compound, composition, preparation method, and application. The aim is to provide a natural extract with whitening, insecticidal, and antibacterial properties.

[0007] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0008] In a first aspect, an alkaloid compound comprising a compound of formula (I) or a medicinal salt or medicinal esterified derivative of formula (I):

[0009]

[0010] The beneficial effects of this invention are:

[0011] (1) This invention is the first to isolate this new alkaloid compound from the tuberous root of the plant Stephania kwangsiensis in Guangxi, with the molecular formula C20H20NO5. + It was named 4,9-dihydroxyl-2,3,10-trimethoxyl berberine.

[0012] (2) The novel alkaloid compounds provided by this invention have been confirmed by in vitro pharmacological experiments to have certain enzyme inhibitory activity, of which IC 50 The value was 1.4758±0.013 mg / mL; it has insecticidal activity, and the killing effect of citrus psyllid at a concentration of 1 mg / mL aqueous solution was 48.70±0.36%; it has antibacterial activity, with a minimum inhibitory concentration (MIC) of 512 μg / mL against Staphylococcus aureus and a minimum inhibitory concentration (MIC) of 1024 μg / mL against beta-hemolytic streptococci.

[0013] In a second aspect, a pharmaceutical composition is characterized in that it comprises the aforementioned alkaloid compound.

[0014] Furthermore, it also includes pharmaceutically acceptable drug excipients and / or carriers. These drug excipients and / or carriers include pharmaceutically acceptable carriers, diluents, fillers, binders, and other excipients, depending on the route of administration and the designed dosage form.

[0015] Furthermore, the dosage form of the pharmaceutical composition is tablets, granules, capsules, nanoparticles, sprays, controlled-release formulations, or sustained-release formulations.

[0016] Furthermore, the actual dosage of the active ingredient in the pharmaceutical composition (4,9-dihydroxyl-2,3,10-trimethoxyl berberine of the present invention) should be determined based on various relevant factors, including the severity of the disease to be treated, the route of administration, the patient's age, sex, and weight. Therefore, the above dosage should not limit the scope of protection of the present invention in any way. The appropriate dosage of the inhibitor can be prescribed in various ways based on factors such as the formulation method, route of administration, the patient's age, weight, sex, condition, diet, administration time, route of administration, excretion rate, and responsiveness. Skilled physicians can usually easily determine the prescription and the dosage that is effective for the desired treatment.

[0017] Furthermore, the following steps are included:

[0018] Step 1: Preparation of primary extract: The raw material of *Dibu Rong* is extracted by immersion in an alcohol-containing aqueous solution to obtain an alcohol-containing extract. The alcohol solution is removed from the alcohol-containing extract (e.g., by vacuum concentration) and then filtered to obtain the primary extract.

[0019] Step 2: Crude component enrichment: The initial extract was purified by macroporous adsorption resin column chromatography, eluted with an alcohol-water solution, and the components containing alkaloids were collected and concentrated to obtain the crude extract.

[0020] Step 3: Preliminary separation and purification: The crude extract was purified sequentially by MCI gel CHP 20P and column chromatography, eluting with an alcohol-water solution. The components containing alkaloids were collected and concentrated to obtain a high-purity extract.

[0021] Step 4: Further separation and purification: The high-purity extract is purified by dextran gel column chromatography, eluted with an alcohol-water solution, and the alkaloid-containing components are collected and concentrated to obtain the alkaloid compounds.

[0022] The aforementioned vacuum concentration is a method of evaporating and drying the solvent by using vacuum to bring it to its boiling point.

[0023] Furthermore, in step 1, the volume fraction of alcohol in the alcohol-containing aqueous solution is 70-75%, such as 70%, 71%, 72%, 73%, 75%, etc., and the weight of the alcohol-containing aqueous solution is 6-10 times that of the insoluble raw material, such as 6 times, 7 times, 8 times, 9 times, 10 times, etc.; the cold soaking extraction time at room temperature is 12-24 hours, such as 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, etc.

[0024] The macroporous adsorption resin column mentioned in step 2 is a DiaionHP-20 macroporous adsorption resin column, which is eluted sequentially with alcohol-containing aqueous solutions of increasing volume fraction (e.g., 0%, 20%, 40%, 60%, 80%, 100%).

[0025] The chromatographic column mentioned in step 3 is a Chromatorex C10 column. 18 The column was eluted sequentially with an increasing volume fraction of alcohol-containing aqueous solution (e.g., 0%, 10%, 20%, 30%, 40%, 50%, 60%).

[0026] The dextran gel column mentioned in step 4 is a Sephadex LH-20 dextran gel column, which is eluted sequentially with an increasing volume fraction of alcohol-containing aqueous solution (e.g., 0%, 10%, 20%, 30%).

[0027] Furthermore, the *Diburong* mentioned in step 1 refers to the tuber of *Diburong* from Guangxi.

[0028] Furthermore, the alcohol in the alcohol-containing aqueous solution mentioned in step 1, step 2, step 3 or step 4 is any one or a mixture of two or more of methanol, ethanol and propanol.

[0029] Thirdly, the application of an alkaloid compound, wherein the alkaloid compound is used to prepare whitening products, insecticidal biological drugs, or antibacterial drugs.

[0030] Furthermore, the aforementioned alkaloid compounds are used as lead compounds for the preparation of skin whitening products, insecticidal biological drugs, or antibacterial drugs.

[0031] The aforementioned insecticides mainly refer to the control of citrus psyllids, while the aforementioned antibacterial agents mainly inhibit Staphylococcus aureus or beta-hemolytic streptococci, etc. Attached Figure Description

[0032] Figure 1 This invention relates to 4,9-dihydroxyl-2,3,10-trimethoxyl berberine. 1 1H NMR (500MHz, CD3OD) spectrum;

[0033] Figure 2 The 4,9-dihydroxyl-2,3,10-trimethoxyl berberine of the present invention 13 C NMR (125MHz, CD3OD) spectrum;

[0034] Figure 3 HSQC spectrum of 4,9-dihydroxyl-2,3,10-trimethoxyl berberine of the present invention;

[0035] Figure 4 The HMBC spectrum of 4,9-dihydroxyl-2,3,10-trimethoxyl berberine of the present invention;

[0036] Figure 5 HR-ESI-MS spectrum of 4,9-dihydroxyl-2,3,10-trimethoxyl berberine of the present invention. Detailed Implementation

[0037] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0038] Example 1

[0039] This embodiment describes the preparation of an alkaloid compound (4,9-dihydroxyl-2,3,10-trimethoxyl berberine), including the following steps:

[0040] 1. Sources of plants

[0041] The tuberous root samples of the plant Stephania kwangsiensis were collected from Gongcheng Yao Autonomous County, Guilin, Guangxi.

[0042] 2. Extraction and Separation

[0043] (1) The dried Guangxi Dibulus tuber was crushed and extracted with 75% ethanol aqueous solution at room temperature for 24 hours. The extract was concentrated under reduced pressure to remove ethanol and then filtered to obtain the initial extract.

[0044] (2) The initial extract in step (1) was separated by a Diaion HP-20 macroporous resin chromatography column (10.0cm×45.0cm), and was eluted with 0%, 20%, 40%, 60%, 80%, and 100% methanol aqueous solution to remove impurities and purify, and then concentrated to obtain the crude extract.

[0045] (3) The crude extract from step (2) was processed using MCI gel CHP 20P and Chromatorex C 18 The extract was purified by column chromatography by eluting with methanol-water solutions of 0%, 10%, 20%, 30%, 40%, 50%, and 60% (v / v), and then concentrated to obtain a high-purity extract.

[0046] (4) The high-purity extract in step (3) was purified by Sephadex LH-20 dextran gel column chromatography, and eluted sequentially with liquid gradients of 0%, 10%, 20%, and 30% by volume. After concentration and methanol crystallization, pharmacologically active alkaloids 4,9-dihydroxyl-2,3,10-trimethoxyl berberine were obtained.

[0047] 3. Identification of new alkaloid compounds

[0048] in, Figure 1 It is compound 4,9-dihydroxyl-2,3,10-trimethoxyl berberine 1 H-NMR (500MHz, CD3OD) spectrum, Figure 2 It is the compound 4,9-dihydroxyl-2,3,10-trimethoxyl berberine. 113 C-NMR (125MHz, CD3OD), Figure 3 This is the HSQC spectrum of compound 4,9-dihydroxyl-2,3,10-trimethoxyl berberine. Figure 4 This is the HMBC spectrum of compound 4,9-dihydroxyl-2,3,10-trimethoxyl berberine. Figure 5 This is the HR-ESI-MS spectrum of compound 4,9-dihydroxyl-2,3,10-trimethoxyl berberine.

[0049] The alkaloid compound of this invention is a reddish-brown solid. It shows a positive reaction with modified bismuth potassium iodide, suggesting that the compound is an alkaloid. Optical rotation [α] 25D -8.3 。 (c=1mg / mL, MeOH), IR(KBr)cm -1:3298 (hydroxyl); 2830 (methoxy); 1632 (double bond); 1586, 1511 (aromatic nucleus); UV spectrum λ max (MeOH): 228 nm; its quasi-molecular ion peak is shown at m / z 354.1375 in HR-ESI-MS [M]. + The information indicates that its molecular formula is C. 20 H 20 NO5 + The degree of unsaturation is 12, suggesting that the structure may contain a benzene ring. 1 The H-NMR spectrum shows that the berberine skeleton is in the δ-ray diffusive region. H 9.31 (1H, s), δ H The two typical double bond signals at 8.20 (1H, s) correspond to carbon signals δ C 147.5 and 121.8, belonging to positions 8 and 13 respectively. In addition, there are 3 aromatic hydrogen protons, δ... H 7.57 (1H, d, J=8.21Hz), δ H 6.97 (1H, d, J = 8.21 Hz), based on chemical shift, coupling constant, and peak shape, indicates that it represents two adjacent hydrogen signals on a benzene ring coupled in an AB system, suggesting the presence of a 1,2,3,4-tetrasubstituted benzene ring unit; δ H The isolated singlet at 7.13 (1H, s) indicates the presence of a 2,3,4,5,6-pentasubstituted benzene ring unit; 13 δ in C-NMR spectrum C Above 100, there are 15 carbon signals, indicating the presence of two benzene rings, one carbon-carbon double bond signal, and one carbon-nitrogen double bond signal. This is based on the chemical shift δ... H 4.61 (2H, d, J = 6.24Hz), δ H 3.14 (2H, t, J = 6.24 Hz) indicates that it consists of two coupled methylene groups, corresponding to a carbon signal of δ. C 55.7, 22.1. In addition, δ H 3.97 (3H, s), δ H 3.90 (3H, s), δ H There are three methyl singlets at 3.86 (3H, s), combined with δ C 61.3, 56.9, and 56.7 confirm the presence of three methoxy groups in the structure. HMBC spectra show δ... C 133.9 and 6.97 (1H, d, J = 8.21 Hz, H⁻¹¹) and δ H 9.31 (1H, s, H-8) is related, therefore δ C 133.9 is attributed to carbon at position 9; δ H9.31 is classified as H-8 primarily due to its association with C-6 (δ). C 55.7) and C-8a(δ C HMBC hydrogen signal correlation at 135.8); δ C 151.7(C-2) and δ H A correlation exists between 3.90 (3H, s) and δ. C 139.4 (C-3) and δ H A correlation exists between 3.86 (3H, s) and δ. C 154.2 (C-10) and δ H The correlation at 3.97 (3H, s) indicates that methoxy groups are substituted at all three positions. δ C There is no correlation between 133.9 (C-9) and the methoxy hydrogen signal, suggesting that this position should be hydroxyl-substituted. δ H 7.13 (1H, s) and δ C 139.4 (C-3), δ C 116.7(C-4a), 124.8(C-14a), δ C 141.7 (C-14) is associated with H-1, δ H 3.14 (2H, t, J = 6.24Hz) and δ C 55.7 (C-6), 116.7 (C-4a), 148.6 (C-4), and 124.8 (C-14a) are associated with H-5, which, combined with the δ in HMQC, indicates a correlation. H 7.13(1H, s, H-1) corresponds to δ C 101.4(C-1), δ H 3.14 (2H, t, J = 6.24 Hz, H⁻⁵) corresponds to δ C 22.1(C-5) corroborates that rings A and B are connected via C-4a and C-14. Furthermore, the HSQC spectrum also shows δ H 6.97 (1H, d, J = 8.21Hz, H⁻¹¹) corresponds to δ C 108.8 (C-11), δ H 7.57 (1H, d, J = 8.21Hz, H⁻¹²) corresponds to δ C 124.4(C-12), δ H 8.20(1H, s, H-13) corresponds to δ C121.8(C-13). The structure of compound SD-1 was determined through spectroscopic data analysis and comparison with similar compounds in the literature, and it was named 4,9-dihydroxyl-2,3,10-trimethoxyl berberine. A search of the CAS-SciFinder database revealed no related reports for this compound, thus confirming it as a novel compound. 1 H-NMR (500MHz, CD3OD), 13 The C-NMR (125MHz, CD3OD) data are shown in the table below.

[0050] Table 1. Compound 4,9-dihydroxyl-2,3,10-trimethoxyl berberine 1 H-NMR data and 13 C-NMR data

[0051]

[0052]

[0053] Based on the comprehensive analysis of the above mass spectrometry, one-dimensional and two-dimensional NMR data, please refer to [link / reference]. Figure 1-5 According to Table 1, the structural formula (Ⅰ) of the compound is derived and named 4,9-dihydroxyl-2,3,10-trimethoxyl berberine.

[0054]

[0055] Example 2:

[0056] This example demonstrates the detection of tyrosinase activity in the alkaloid compound 4,9-dihydroxyl-2,3,10-trimethoxyl berberine.

[0057] Levodopa (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) was used as the substrate (1 mg / mL), and kojic acid (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) was used as the positive control. The sample mentioned below is the alkaloid compound 4,9-dihydroxyl-2,3,10-trimethoxyl berberine of this invention. The samples were divided into three groups: Sample A (20 μL sample or kojic acid + 10 μL tyrosinase), Sample A control group (20 μL sample or kojic acid + 10 μL PBS), Blank Group B (20 μL PBS + 10 μL tyrosinase), and Blank Control Group B (30 μL PBS). These were added sequentially to 96-well plates and incubated at 37°C for 10 min. Then, 40 μL of levodopa was added to each well, and the plates were incubated at 37°C for 5 min. The absorbance was measured at 475 nm. Each group was tested in triplicate. The IC50 value was calculated based on the tyrosinase activity inhibition rate. 50 The value was [value missing]. Tyrosinase was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0058] Tyrosinase inhibition rate (%) = [1 - (Sample A group - Sample A control group) / (B blank group - B blank control group)] × 100%.

[0059] Experimental results: The alkaloid compound 4,9-dihydroxyl-2,3,10-trimethoxylberberine of this invention has an effect on tyrosinase IC50. 50 Value: 1.4758±0.013mg / mL.

[0060] Example 3:

[0061] This example demonstrates the detection of the killing activity of the alkaloid compound 4,9-dihydroxyl-2,3,10-trimethoxyl berberine against citrus psyllids.

[0062] Citrus trees showing symptoms of Huanglongbing (HLB) were selected from the orchard. Several shoots from each tree were sampled to examine infested leaves. Both adult and larval stages of the citrus psyllid were present, and the number of live psyllids was recorded. The initial psyllid population was assessed before and again 6 days after application. Both compounds were dissolved in a small amount of methanol, diluted with water to 1000 mg / kg, and evenly sprayed onto leaves and branches using a handheld micro-sprayer. The experiment was repeated three times. A control group was prepared using water, with the remaining steps the same.

[0063] Mortality rate = (Number of live insects in the treated area before treatment / Number of live insects in the treated area after treatment) × 100%; Corrected mortality rate = [(Mortality rate of the treated group - Mortality rate of the control group) / (1 - Mortality rate of the control group)] × 100%

[0064] Experimental results: The mortality rate of the blank control was 8.96±0.21%; the mortality rate of the compound was 48.70±0.36%, and the corrected mortality rate was 43.38%.

[0065] As can be seen from the above experimental results, the alkaloid compound 4,9-dihydroxyl-2,3,10-trimethoxyl berberine of the present invention has a certain inhibitory effect on citrus psyllids, providing new conditions for the development of novel natural insecticides.

[0066] Example 4:

[0067] This example demonstrates the in vitro inhibitory activity of the alkaloid compound 4,9-dihydroxyl-2,3,10-trimethoxyl berberine against Staphylococcus aureus and beta-hemolytic streptococci.

[0068] All bacterial strains used in the experiment were standard strains, purchased from Shanghai Baozang Microbial Co., Ltd.

[0069] Preparation of bacterial suspension: The sample was prepared using DMSO as a solvent to a concentration of 51.2 mg / mL, sealed, and refrigerated until use. The preparation of MH agar and MH broth media followed the relevant testing standards and culture medium instructions of the National Clinical and Laboratory Standards Institute (NCCLS). Staphylococcus aureus and beta-hemolytic streptococci were inoculated separately into MH broth media and incubated at 37°C for 24 hours. Then, they were cultured on a shaker to prepare bacterial suspensions, which were then diluted to 10⁻⁶. 3 / mL~10 4 / times, for backup.

[0070] Determination of minimum inhibitory concentration (MIC):

[0071] MIC was determined using the 96-well plate azuril colorimetric method: After sterilizing all tools, the plates were placed in a clean bench. Under aseptic conditions, the sample solution was diluted to 2048 μg / mL with broth. A sterile 96-well plate was used, and 100 μL of MH broth was added to each well. For the first row of wells, 100 μL of the 2048 μg / mL sample solution was added and mixed thoroughly. Then, 100 μL of the mixture was added to the second row, and so on, decreasing the sample concentration from top to bottom (discarding the last 100 μL). A total of five concentrations were determined. Finally, 100 μL of MH bacterial broth was added to each well. For the positive control group, the sample solution was replaced with 100 μL of kanamycin sulfate (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) or ampicillin (purchased from Shanghai Yuanye Biotechnology Co., Ltd.). For the negative control group, the sample solution was replaced with 100 μL of broth. Place the sample in a 37℃ incubator and incubate for 18-20 hours. Then add 10 μL of resazurine chromogenic agent and observe the results. Pink indicates bacterial growth, and blue indicates inhibition of bacterial growth. Determine the minimum inhibitory concentration (MIC) of the sample.

[0072] Experimental results: The MIC for Staphylococcus aureus was 512 μg / mL, and the MIC for beta-hemolytic streptococci was 1024 μg / mL. These results indicate that 4,9-dihydroxyl-2,3,10-trimethoxyl berberine exhibits good inhibitory activity against Staphylococcus aureus, thus it holds promise as a compound or lead compound for developing new antibacterial drugs.

[0073] In summary, the novel alkaloid compounds provided by this invention have been confirmed by in vitro pharmacological experiments to possess certain enzyme inhibitory activity, with an IC50 value of 1.4758±0.013 mg / mL; insecticidal activity, with a killing effect of 48.70±0.36% against citrus psyllids; and antibacterial activity, with a minimum inhibitory concentration (MIC) of 512 μg / mL against Staphylococcus aureus and 1024 μg / mL against beta-hemolytic streptococci at a concentration of 1 mg / mL in aqueous solution.

[0074] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. The application of an alkaloid compound, characterized in that, The alkaloid compound of formula (I) or the medicinal salt of formula (I) is used to prepare an insecticidal biological drug; the insect is the orange psyllid; The alkaloid compounds include compounds as shown in formula (Ⅰ): 。 2. The application of the alkaloid compound according to claim 1, characterized in that, The method for preparing the alkaloid compound includes the following steps: Step 1: Preparation of primary extract: The raw material of *Dibu Rong* is extracted by immersion in an alcohol-containing aqueous solution to obtain an alcohol-containing extract. After removing the alcohol solution from the alcohol-containing extract, it is filtered to obtain the primary extract. Step 2: Crude component enrichment: The initial extract was purified by macroporous adsorption resin column chromatography, eluted with an alcohol-water solution, and the components containing alkaloids were collected and concentrated to obtain the crude extract. Step 3: Preliminary separation and purification: The crude extract was purified sequentially by MCIgelCHP 20P column chromatography, with elution using an alcohol-water solution. The components containing alkaloids were collected and concentrated to obtain a high-purity extract. Step 4: Further separation and purification: The high-purity extract is purified by dextran gel column chromatography, eluted with an alcohol-water solution, and the alkaloid-containing components are collected and concentrated to obtain the alkaloid compounds.

3. The application of the alkaloid compound according to claim 2, characterized in that, The alcohol-containing aqueous solution in step 1 has a volume fraction of 70-75%, and the weight of the alcohol-containing aqueous solution is 6-10 times that of the insoluble raw material; the cold maceration extraction time at room temperature is 12-24 hours; The macroporous adsorption resin column mentioned in step 2 is a DiaionHP-20 macroporous adsorption resin column, which is eluted sequentially with alcohol-containing aqueous solutions of increasing volume fraction. The chromatographic column used in step 3 is a Chromatorex C18 column, which is eluted sequentially with an increasing volume fraction of alcohol-containing aqueous solution. The dextran gel column mentioned in step 4 is a Sephadex LH-20 dextran gel column, which is eluted sequentially with an increasing volume fraction of alcohol-containing aqueous solution.

4. The application of the alkaloid compound according to claim 2, characterized in that, The "Dibu Rong" mentioned in step 1 refers to the tuberous root of the Guangxi Dibu Rong plant.

5. The application of the alkaloid compound according to claim 2, characterized in that, The alcohol in the alcohol-containing aqueous solution mentioned in step 1, step 2, step 3 or step 4 is any one or a mixture of two or more of methanol, ethanol and propanol.

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