Novel indole alkaloids from Gelsemium elegans, their preparation methods and applications

Abstract

This invention discloses novel indole alkaloids from *Gelsemium elegans*, their preparation methods, and applications, belonging to the field of pharmaceutical technology. Two novel indole alkaloids, 23-methylgelsecrotonidine and 14-hydroxy-23-methylgelsecrotonidine, were extracted and isolated from *Gelsemium elegans* Benth. (family Loganiaceae), possessing the structure shown in formula (I). They were prepared by sequentially employing methods including ethanol reflux extraction, acid-base precipitation, ethyl acetate extraction, rapid column chromatography, pH-zone purification countercurrent chromatography, and conventional column chromatography. The novel indole alkaloids provided by this invention significantly inhibited LPS-induced iNOS overexpression in RAW264.7 inflammatory cells, reduced NO release and the production of inflammatory factors TNF-α and IL-6, exhibiting excellent anti-inflammatory and immunomodulatory activity, and can be used for the development of anti-inflammatory and immunomodulatory drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to novel indole alkaloid compounds from *Gelsemium elegans*, a plant in the Loganiaceae family, their preparation methods, and their applications in the preparation of anti-inflammatory and immunomodulatory drugs. Background Technology

[0002] Gelsemium elegans Benth., a perennial evergreen climbing vine belonging to the genus Gelsemium in the family Loganiaceae, is also known as Gelsemium elegans, Gelsemium elegans, and Gelsemium elegans. It is a traditional medicinal plant, and the entire plant can be used medicinally. Gelsemium elegans has diverse chemical components, with the main active ingredients being indole alkaloids. Several patents have been published regarding its application in the preparation of drugs for treating rheumatoid arthritis (patent publication number: CN102293768B), drugs for treating chronic pain (patent publication number: CN101322705B), and the treatment of amyloidosis and / or tau lesions-related diseases or conditions (patent publication number: CN109953991B).

[0003] In addition to gelseminalis, patent application CN113185528A discloses the selective anti-osteoclast activity of gelseminalis alkaloid 14-hydroxygelsenicine and its pharmaceutical application in the prevention and treatment of bone metabolic diseases; patent applications CN116059200A and CN114246863A disclose the application of gelseminalis chlorophyllin in the preparation of drugs for treating arthritis and in products for inhibiting osteoporosis, respectively. All of the above applications are closely related to the anti-inflammatory and immunomodulatory effects of gelseminalis indole alkaloids.

[0004] Inflammation is a defensive response of the body to stimuli. A moderate inflammatory response helps the body fight off external pathogens; however, a disordered and persistent inflammatory response leads to tissue damage and organic lesions, inducing various inflammatory diseases. Modern research shows that inflammation is associated with many diseases. Various chronic diseases, including rheumatoid arthritis and neurodegenerative diseases, are caused by long-term inflammatory responses. Inhibiting inflammatory responses is the first step in treating many diseases, but persistent inflammation easily leads to autoimmune diseases. Therefore, developing natural anti-inflammatory and immunomodulatory drugs with fewer side effects is essential for the treatment of these diseases. The aforementioned reports indicate that Gelsemium elegans indole alkaloids have great development potential and research value. Therefore, discovering indole alkaloids with good anti-inflammatory and immunomodulatory activity and novel structures from Gelsemium elegans is of great significance for new drug development. Summary of the Invention

[0005] The present invention aims to provide novel indole alkaloid compounds obtained from Gelsemium elegans and methods for their preparation, as well as the application of these compounds in the preparation of anti-inflammatory and immunomodulatory drugs.

[0006] The novel indole alkaloid compound provided by this invention has the molecular formula C0. 23 H 26 N2O5 and C 23 H 26 N₂O₆, named 23-methylgelsecrotonidine and 14-hydroxy-23-methylgelsecrotonidine respectively, has the following chemical structural formulas:

[0007]

[0008] The novel indole alkaloids described in this invention can form salts with acids to generate their pharmaceutically acceptable salts.

[0009] The pharmaceutically acceptable salts described in this invention are selected from hydrochloride, sulfate, hydrobromide, hydroiodide, nitrate, hydrogen sulfate, phosphate, acid phosphate, citrate, acetate, oxalate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, hydrogen tartrate, ascorbate, succinate, maleate, gentianate, fumarate, gluconate, glucuronide, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, or pyrazocyanate.

[0010] This invention also provides a method for preparing the above-mentioned novel indole alkaloid compounds, comprising the following steps:

[0011] 1) Take the dried stem of Gelsemium elegans Benth., a plant of the Loganiaceae family, crush it, and extract it by reflux with ethanol. Concentrate the extract to obtain Gelsemium elegans extract, remove impurities by alcohol-water method, acid dissolution and alkali precipitation, and concentrate the ethyl acetate extract to obtain total alkaloids of Gelsemium elegans.

[0012] 2) The total alkaloids of Gelsemium elegans were subjected to rapid column chromatography and eluted with a dichloromethane-methanol system to obtain fraction AC.

[0013] 3) The component A obtained in step 2) was separated by pH-zone purification countercurrent chromatography and detected by high performance liquid chromatography to obtain subcomponents I-VII.

[0014] 4) The subfraction VII obtained in step 3) was purified by conventional column chromatography, eluted with petroleum ether-ethyl acetate system, and detected by high performance liquid chromatography to obtain the new indole alkaloids 23-methylgelsecrotonidine and 14-hydroxy-23-methylgelsecrotonidine.

[0015] The method for preparing the novel indole alkaloid compound of the present invention, wherein:

[0016] In step 1), the ethanol concentration is 70-95%; the reflux extraction is carried out at 70-90℃ with stirring for 6-8 hours, and the extraction is performed 3-4 times; the acid dissolution and alkali precipitation is carried out by adjusting the pH of the system to 1-3 with dilute hydrochloric acid, and then adding NaOH aqueous solution to the acidic aqueous phase to adjust the pH of the system to 9-11.

[0017] In step 2), the weight ratio of silica gel to sample in the rapid column chromatography is 5-10:1; the elution volume ratio of the dichloromethane-methanol system is 80-1:1.

[0018] In step 3), the solvent system used in the pH zone purification countercurrent chromatography is methyl tert-butyl ether-n-butanol-acetonitrile-water (4:0:1:5-2:0:2:3, V:V), the upper phase is adjusted to pH=9-11 as the stationary phase, and the lower phase is adjusted to pH=1-3 as the mobile phase.

[0019] In step (4), the weight ratio of silica gel to sample in the conventional column chromatography is 30-100:1; the elution volume ratio of the petroleum ether-ethyl acetate system is 20-1:1.

[0020] The present invention also includes the use of the novel indole alkaloids and their pharmaceutically acceptable salts in the preparation of anti-inflammatory and immunomodulatory drugs.

[0021] The novel indole alkaloid compounds described in this invention can be formulated into pharmaceutically acceptable solid or liquid dosage forms by adding pharmaceutically acceptable excipients.

[0022] The beneficial effects of this invention are:

[0023] 1. This invention discloses two novel indole alkaloids, 23-methylgelsecrotonidine and 14-hydroxy-23-methylgelsecrotonidine, obtained from Gelsemium elegans. The structures of these compounds were confirmed by mass spectrometry, carbon spectroscopy, proton spectroscopy, and two-dimensional nuclear magnetic resonance spectroscopy. A search on SciFinder showed that these compounds were not yet indexed, thus identifying them as novel compounds. These new indole alkaloids enrich the structural diversity of this class of compounds and provide a material basis for their pharmacodynamic studies.

[0024] 2. This invention provides a method for preparing novel indole alkaloids 23-methylgelsecrotonidine and 14-hydroxy-23-methylgelsecrotonidine. After extracting the total alkaloids from Gelsemium elegans, the method sequentially employs rapid column chromatography, pH-zone purification countercurrent chromatography, and conventional column chromatography to obtain the target compounds. This method offers good controllability and reproducibility, making it suitable for the separation and purification of trace compounds.

[0025] 3. The novel indole alkaloid compounds 23-methylgelsecrotonidine and 14-hydroxy-23-methylgelsecrotonidine, and their pharmaceutically acceptable salts, described in this invention, can significantly inhibit the overexpression of iNOS in LPS-induced RAW264.7 inflammatory cells, reduce NO release and the production of inflammatory factors TNF-α and IL-6, exhibiting good anti-inflammatory and immunomodulatory activity. They have the potential to be further developed into anti-inflammatory and immunomodulatory drugs, providing clues for new drug development. Attached Figure Description

[0026] Figure 1 This is a countercurrent chromatogram of the pH zone purification of Gelsemium alkaloid component A in Example 1;

[0027] Figure 2 The high-performance liquid chromatogram of subcomponent VII in Example 1;

[0028] Figure 3 The HRMS spectrum of the novel compound 23-methylgelsecrotonidine in Example 2;

[0029] Figure 4 The novel compound 23-methylgelsecrotonidine in Example 2 1 H-NMR spectrum (600MHz, CDCl3);

[0030] Figure 5 The novel compound 23-methylgelsecrotonidine in Example 2 13 C-NMR spectrum (150MHz, CDCl3);

[0031] Figure 6 The HSQC spectrum (600 MHz, CDCl3) of the novel compound 23-methylgelsecrotonidine in Example 2;

[0032] Figure 7 The novel compound 23-methylgelsecrotonidine in Example 2 1 H-1 H COSY spectrum (600MHz, CD Cl3);

[0033] Figure 8 The HMBC spectrum (600 MHz, CDCl3) of the novel compound 23-methylgelsecrotonidine in Example 2;

[0034] Figure 9 The HRMS spectrum of the novel compound 14-hydroxy-23-methylgelsecrotonidine in Example 2;

[0035] Figure 10 The novel compound 14-hydroxy-23-methylgelsecrotonidine in Example 2 1 H-NMR spectrum (600MHz, CDCl3);

[0036] Figure 11 The novel compound 14-hydroxy-23-methylgelsecrotonidine in Example 2 13 C-NMR spectrum (150 M Hz, CDCl3);

[0037] Figure 12 The HSQC spectrum (600 MHz, CDCl3) of the novel compound 14-hydroxy-23-methylgelsecrotonidine in Example 2;

[0038] Figure 13 The novel compound 14-hydroxy-23-methylgelsecrotonidine in Example 2 1 H- 1 H COSY spectrum (600MHz, CDCl3);

[0039] Figure 14 The HMBC spectrum (600 MHz, CDCl3) of the novel compound 14-hydroxy-23-methylgelsecrotonidine in Example 2;

[0040] Figure 15 Key to the novel compounds 23-methylgelsecrotonidine and 14-hydroxy-23-methylgelsecrotonidine in Example 2 1 H- 1 H COSY and HMBC correlation and CD spectrum;

[0041] Figure 16 The effects of the novel compounds 23-methylgelsecrotonidine and 14-hydroxy-23-methylgelsecrotonidine in Example 3 on the viability of RAW264.7 cells;

[0042] Figure 17 The effects of the novel compounds 23-methylgelsecrotonidine (compound 1) and 14-hydroxy-23-methylgelsecrotonidine (compound 2) in Example 4 on LPS-induced NO release (A) and iNOS expression level (BC) in RAW264.7 cells were investigated.

[0043] Figure 18 The effects of the novel compounds 23-methylgelsecrotonidine (compound 1) and 14-hydroxy-23-methylgelsecrotonidine (compound 2) in Example 5 on the production of TNF-α (A) and IL-6 (B) in LPS-induced RAW264.7 cells. Detailed Implementation

[0044] The present invention will be further described below with reference to specific embodiments.

[0045] Example 1: Preparation method of the new compounds 23-methylgelsecrotonidine and 14-hydro-23-methylgelsecrotonidine, the specific steps are as follows:

[0046] 1) 500 kg of dried stem powder of Gelsemium elegans was extracted three times with 95% ethanol at 70-75℃ for 8 hours each time. The filtrate was concentrated under reduced pressure to obtain a dark brown extract. After removing water-insoluble impurities from the extract by alcohol-water method, the pH of the system was adjusted to 1-3 with 5% hydrochloric acid. The acidic aqueous phase was extracted with ethyl acetate to remove lipids and pigments. Then, the pH of the system was adjusted to 9-11 with 5% sodium hydroxide aqueous solution. The extract was then extracted with ethyl acetate again. The extract was concentrated to obtain total alkaloids of Gelsemium elegans (2715.5 g).

[0047] 2) The total alkaloids of Gelsemium elegans obtained in step 1) were crudely separated by rapid column chromatography. The ratio of silica gel to sample was 8:1 (W:W), and the eluent was dichloromethane and methanol in a ratio of 80:1:1 (V:V). TLC was monitored in real time. Using Gelsemium elegans as a reference, alkaloid fractions with lower polarity than Gelsemium elegans were collected, combined, and dried by rotary evaporation to obtain fraction A (660.2 g).

[0048] 3) Component A obtained in step 2) was separated using pH-zone purification countercurrent chromatography. The solvent system was methyl tert-butyl ether-n-butanol-acetonitrile-water (4:0:1:5, V:V). The upper phase was adjusted to pH=9 with triethylamine to serve as the stationary phase; the lower phase was adjusted to pH=2 with concentrated hydrochloric acid to serve as the mobile phase. The stationary phase was pumped into the high-speed countercurrent chromatograph at 30 mL / min until full, the main unit was rotated forward at 850 rpm, the temperature of the constant temperature circulator was 25℃, and the wavelength of the UV detector was 254 nm. 5.0 g of component A was dissolved in 10 mL of the stationary phase and injected through a six-way valve. The mobile phase was then pumped in at a flow rate of 6.0 mL / min. The chromatogram was acquired by the chromatography workstation, and the sample was collected by the automatic fraction collector. Separation was completed when the pH of the pumped fraction reached 3. The pH-zone purification countercurrent chromatogram of component A is shown below. Figure 1 Each fraction was analyzed by high-performance liquid chromatography (HPLC) to obtain subfractions I-VII. The HPLC chromatogram of subfraction VII is shown below. Figure 2 .

[0049] 4) Repeat the above steps, combine the subfractions VII obtained from multiple separations, purify by conventional column chromatography with gradient elution in a petroleum ether-ethyl acetate system (20:1-1:2, V:V), detect by high performance liquid chromatography, combine identical components, concentrate under reduced pressure, and freeze-dry to obtain 28.0 mg of compound 23-methylgelsecrotonidine with a purity of 95.24%; and 85.1 mg of compound 14-hydro-23-methylgelsecrotonidine with a purity of 95.44%.

[0050] Example 2: Structural identification of the new compounds 23-methylgelsecrotonidine and 14-hydro-23-methylgelsecrotonidine

[0051] Compound 23-methylgelsecrotonidine is a brown powder with the chemical formula C. 23 H 26 N₂O₅, the quasi-molecular ion peak is m / z 411.19061 [M+H] + One-dimensional NMR and two-dimensional carbon NMR spectra show that its structure is similar to gelsecrotonidine, the difference being that the terminal structure of the monoterpene moiety is an ethyl ester. A search for this structure on SciFinder showed that it is not yet indexed, therefore it is identified as a new compound. The spectrum is shown below. Figure 3-8 , 15.

[0052] Compound 14-hydro-23-methylgelsecrotonidine is a brown powder with the chemical formula C. 23 H 26N₂O₆, quasi-molecular ion peak is 427.18555 [M+H] + One-dimensional NMR and two-dimensional carbon NMR spectra show that its structure is similar to that of 14-hydro-gelsecrotonidine, the difference being that the terminal structure of the monoterpene moiety is an ethyl ester. A search for this structure on SciFinder showed that it is not yet indexed, therefore it is identified as a new compound. The spectrum is shown below. Figure 9-15 .

[0053] Table 1. 1H NMR (600 MHz, CDCl3) and 150 MHz (CDCl3) spectra of the new compounds 23-methylgelsecrotonidine and 14-hydro-23-methylgelsecrotonidine.

[0054]

[0055] Example 3: Effects of the novel compounds 23-methylgelsecrotonidine and 14-hydroxy-23-methylgelsecrotonidine on the viability of RAW264.7 cells

[0056] Collect cells in the logarithmic growth phase, count them, and adjust the cell suspension concentration to 1×10⁻⁶. 5 Cells / mL. Seeds were added to 96-well plates, with 100 μL of cell suspension added to each well, resulting in a cell count of 1 × 10⁶ cells per well. 4 The experiment was divided into two groups: a control group and treatment groups with different concentrations of 23-methylgelsecrotonidine and 14-hydroxy-23-methylgelsecrotonidine, with 5 replicates per group. Cells were cultured at 37℃ and 5% CO2 for 24 hours until adherence. The supernatant was discarded. The control group was added to 100 μL of medium containing 0.1% DMSO and 1% FBSDMEM, while the treatment groups were added to 100 μL of medium containing different concentrations of 23-methylgelsecrotonidine and 14-hydroxy-23-methylgelsecrotonidine. After 24 hours, the old medium was discarded, and 100 μL of prepared medium containing 10% CCK-8 was added to each well. Cells were cultured for another 1 hour, and the OD value was measured at 450 nm. Cell viability was calculated using the following formula: Cell viability = [(OD value - ... 实验孔 -OD 空白孔 ) / (OD 对照孔 -OD 空白孔 )]×100%, results are shown in Figure 16The results showed that the new compounds 23-methylgelsecrotonidine and 14-hydroxy-23-methylgelsecrotonidine had no effect on the viability of RAW264.7 cells at a concentration of 100 μM. Example 4: The new compounds 23-methylgelsecrotonidine and 14-hydroxy-23-methylgelsecrotonidine reduced the release of the inflammatory mediator NO in RAW264.7 cells by inhibiting LPS-induced iNOS overexpression.

[0057] RAW264.7 cells in logarithmic growth phase were collected, counted, and seeded into 96-well plates at a density of 5 × 10⁶ cells per well. 4 The experiment was conducted in three groups: a control group, an LPS model group, an indomethacin positive control group, and drug administration groups containing different concentrations of 23-methylgelsecrotonidine and 14-hydroxy-23-methylgelsecrotonidine, with three replicates per group. After incubation at 37°C and 5% CO2 for 24 hours, the supernatant was discarded. The control group and LPS model group were given 100 μL of DMEM high-glucose medium containing 0.1% DMSO and 1% FBS. The positive control group was given 100 μL of 20 μM indomethacin, and the drug administration groups were given 100 μL of different concentrations of the novel compounds 23-methylgelsecrotonidine and 14-hydroxy-23-methylgelsecrotonidine. After 2 hours of incubation, 100 μL of LPS was added to the model group and the drug-treated group to bring the final concentration to 1 μg / mL. The control group was incubated with 100 μL of DMEM high-glucose medium. After 24 hours of further incubation, 50 μL of cell supernatant from each well was transferred to another 96-well plate. 50 μL each of Griess Reagent I and II were added sequentially, and the plate was incubated in the dark for 10 minutes. The OD value was measured at 540 nm. The NO concentration was calculated using a standard curve based on NaNO2, and the IC50 was calculated by fitting the data using GraphPad Prism 9 software. 50 Subsequently, the cell surface was washed with PBS, and 150 μL of lysis buffer containing 1% PMSF was added for thorough lysis for 30 min. The cell lysate was collected in pre-chilled 1.5 mL centrifuge tubes and centrifuged at 12000 rpm and 4 °C for 20 min. The supernatant was used to determine the protein concentration using the BCA method, and the iNOS protein expression level was detected by Western blotting. The effects of the new compound on LPS-induced NO release and iNOS expression levels in RAW 264.7 cells are described in [the table below]. Figure 17The results showed that both 23-methylgelsecrotoni dine and 14-hydroxy-23-methylgelsecrotoni dine reduced LPS-induced NO release in RAW264.7 cells, and the IC50 value was [not specified]. 50 The effective concentrations were 35.50±9.81 and 72.96±21.17 μM, respectively; both significantly inhibited the overexpression of iNOS protein.

[0058] Example 5: The novel compound 23-methylgelsecrotonidine inhibits the production of inflammatory factors TNF-α and IL-6 in LPS-induced RAW264.7 inflammatory cells.

[0059] RAW264.7 cells in logarithmic growth phase were collected, counted, and seeded into 96-well plates at a density of 5 × 10⁶ cells per well. 5 Cells were cultured at 37℃ in a 5% CO2 incubator for 24 h. The experiment included a control group, an LPS model group, an indomethacin positive control group (20 μM), and different concentrations of 23-methylgelsecrotonidine and 14-hydroxy-23-methylgelsecrotonidine (25, 50, 100 μM) administration groups, with three replicates per group. After 2 h of pretreatment in the positive control and administration groups, the LPS model group, positive control group, and administration groups were added to the LPS model group, positive control group, and administration groups, and cultured for another 24 h. Cell supernatants were collected, centrifuged at 12000 rpm at 4℃ for 20 min to remove precipitates, and stored at -80℃. The levels of TNF-α and IL-6 in the cell supernatant were detected using an ELISA kit. The effect of the new compounds on LPS-induced TNF-α and IL-6 production in RAW264.7 cells is described in [reference needed]. Figure 18 The results showed that 50-100 μM 23-methylgelsecrotonidine and 25-100 μM 14-hydroxy-23-methylgelsecrotonidine could inhibit the production of TNF-α and IL-6 in a dose-dependent manner, and 23-methylgelsecrotonidine had stronger anti-inflammatory and immunomodulatory activity than 14-hydroxy-23-methylgelsecrotonidine.

[0060] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. An indole alkaloid compound from Gelsemium elegans, characterized in that, Its chemical structure is as follows: 。 2. The use of the indole alkaloids and their pharmaceutically acceptable salts as described in claim 1 in the preparation of anti-inflammatory drugs.

3. The application of the indole alkaloids and their pharmaceutically acceptable salts according to claim 2 in the preparation of anti-inflammatory drugs, characterized in that: The pharmaceutically acceptable salts are selected from hydrochloride, sulfate, hydrobromide, hydroiodide, nitrate, hydrogen sulfate, phosphate, acid phosphate, citrate, acetate, oxalate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, hydrogen tartrate, ascorbate, succinate, maleate, gentianate, fumarate, gluconate, glucuronide, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, or pyrazocyanate.

4. The application of the indole alkaloids and their pharmaceutically acceptable salts according to claim 2 in the preparation of anti-inflammatory drugs, characterized in that: Anti-inflammatory drugs are formulated into pharmaceutically acceptable solid or liquid dosage forms using pharmaceutically acceptable excipients.

5. The method for preparing the indole alkaloid compound as described in claim 1, characterized in that, Includes the following steps: 1) Take the dried stem of Gelsemium elegans, a plant of the Loganiaceae family, crush it, and extract it by reflux with ethanol. Concentrate the extract to obtain Gelsemium elegans extract, remove impurities by alcohol-water method, acid dissolution and alkali precipitation, and concentrate the ethyl acetate extract to obtain total alkaloids of Gelsemium elegans. 2) The total alkaloids of Gelsemium elegans were subjected to rapid column chromatography and eluted with a dichloromethane-methanol system to obtain fraction AC; 3) Component A obtained in step 2) is separated by pH-zone purification countercurrent chromatography and detected by high performance liquid chromatography to obtain subcomponents I-VII; the solvent system of the pH-zone purification countercurrent chromatography is methyl tert-butyl ether-n-butanol-acetonitrile-water; 4) The subfraction VII obtained in step 3) was purified by conventional column chromatography, eluted with petroleum ether-ethyl acetate system, and detected by high performance liquid chromatography to obtain indole alkaloids 23-methylgelsecrotonidine and 14-hydroxy-23-methylgelsecrotonidine.

6. The method for preparing the indole alkaloid compound according to claim 5, characterized in that: In step 1), the ethanol concentration is 70-95%; the reflux extraction is carried out by stirring at 70-90℃ for 6-8 hours, and the extraction is repeated 3-4 times; the acid dissolution and alkali precipitation is carried out by adjusting the pH of the system to 1-3 with dilute hydrochloric acid, and then adding sodium hydroxide aqueous solution to adjust the pH of the system to 9-11.

7. The method for preparing the indole alkaloid compound according to claim 5, characterized in that: In step 2), the weight ratio of silica gel to sample in the rapid column chromatography is 5-10:1; the elution volume ratio of the dichloromethane-methanol system is 80-1:

1.

8. The method for preparing the indole alkaloid compound according to claim 5, characterized in that: In step 3), the methyl tert-butyl ether-n-butanol-acetonitrile-water are in a volume ratio of 4:0:1:5 - 2:0:2:3, with the upper phase adjusted to pH 9-11 as the stationary phase and the lower phase adjusted to pH 1-3 as the mobile phase.

9. The method for preparing the indole alkaloid compound according to claim 5, characterized in that: In step 4), the weight ratio of silica gel to sample in the conventional column chromatography is 30-100:1; the elution volume ratio of the petroleum ether-ethyl acetate system is 20-1:1.

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