Escherichia coli recombinant engineering bacterium and application thereof
By constructing a Fosmid library of human intestinal microorganisms and screening out E. coli F10 with antibacterial activity, the problem of difficulty in efficient isolation of intestinal microorganisms and their metabolites in the prior art is solved, and the efficient preparation of indole compounds is achieved, which has the advantages of green environmental protection and high product purity.
Patent Information
- Application Number
- CN202411617982.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-13
- Publication Date
- 2025-02-25
AI Technical Summary
It is difficult for the existing technology to efficiently discover and isolate intestinal microorganisms and their metabolites, especially low-abundance strains and anaerobic bacteria, which seriously restricts the basic theoretical research and industry development of intestinal microorganisms.
By constructing a Fosmid library of human intestinal microorganisms, and using E. coli E. coli BL21 as the host bacteria, the Fosmid library plasmid DNA was introduced, and E. coli F10 with antibacterial activity was screened out by plate diffusion method, which was used for fermentation and culture and isolation and purification of indole compounds.
The production of indole-3-formaldehyde and indole trimers during the fermentation process of E. coli F10 provides a new green and environmentally friendly preparation method, with the advantages of simple reaction conditions, few by-products and high product purity.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and specifically relates to a recombinant engineering bacterium of Escherichia coli and an application thereof. Background Art
[0002] Microbial natural products have always been an important source for drug development, and are still the main source of new natural products. Among them, polyketides and non-ribosomal peptides are a class of secondary metabolites produced by microorganisms. They are widely available in nature and are an important part of natural products. These compounds have good medicinal value and other important physiological functions, such as the anti-tumor drug azithromycin, the antibacterial drug tetracycline, and cephalosporin. Traditional natural products are generally derived from terrestrial and marine microorganisms. With the in-depth research on these microorganisms and the increase in the base of microbial natural products over the years, the discovery of novel natural products has become increasingly difficult. How to more efficiently discover natural products with new sources and new structures has become a key issue in the study of microbial natural products. In recent years, with the rapid development of intestinal microbiome, intestinal microbial metabolic small molecules are closely related to the balance and homeostasis of the body. At present, the isolation methods of intestinal microorganisms include the following: 1. Gradient dilution plate spreading method: the microorganisms are placed in a specific culture medium for separation and purification, and the number of microorganisms is calculated by methods such as multiple dilution and counting; 2. Microfluidics-based high-throughput separation and culture method: single microorganisms are cultured through single cells in microdroplets, and low-abundance strains are effectively separated.
[0003] In view of the advantages of gradient dilution plate method such as diversity, strong operability and scalability, this traditional separation and culture method is still widely used. However, the traditional dilution plate method has a large overall workload, is time-consuming and labor-intensive, has a long culture cycle, and cannot culture anaerobic bacteria. The microfluidics-based culture method can maintain an anaerobic or low-level oxygen content environment and can be used for the culture of anaerobic microorganisms, but the limiting dilution method used in the microfluidics-based culture process is very unfavorable for the single microbial culture of symbiotic microorganisms and is prone to losing low-abundance strains during the dilution process. According to the latest report on the unified genome of the human gastrointestinal tract, more than 70% of intestinal microbial species have not been cultured (Almeida A, et al. Nature Biotechnology, 2021, 39 (1): 105-114.), which seriously restricts the basic theoretical research and industry development of intestinal microorganisms. Therefore, new technical means are needed for the separation of intestinal microorganisms and their metabolites.
[0004] With the development of high-throughput sequencing or next-generation sequencing technology, microbial research has begun to use amplicon sequencing and metagenomic sequencing. These sequencing methods allow the study of microorganisms to bypass the isolation and cultivation process of microorganisms, thereby providing a way to study microorganisms that cannot be isolated and cultured. To this end, the inventors explored the functional components produced by intestinal microorganisms at the level of intestinal microbial functional genomes through the construction and application of intestinal microbial metagenomic DNA libraries. Summary of the invention
[0005] The purpose of the present invention is to provide a recombinant engineering bacterium of Escherichia coli and its application.
[0006] To achieve the purpose of the invention, the technical solution adopted by the present invention is as follows:
[0007] The first aspect of the present invention provides a recombinant engineered Escherichia coli bacterium, wherein the recombinant engineered Escherichia coli bacterium is Escherichia coli F10, whose Latin name is Escherichia coli F10, which is preserved in the China Center for Type Culture Collection, located in Wuhan, China, with a preservation number of CCTCC NO: M2024964 and a preservation date of May 16, 2024.
[0008] The second aspect of the present invention provides a method for constructing the Escherichia coli recombinant engineering bacteria described in the first aspect. The method for constructing the Escherichia coli F10 is as follows: extracting the DNA of the plasmid in the constructed Fosmid library, using Escherichia coli BL21 as the host bacteria, introducing the extracted Fosmid library plasmid DNA into the host bacteria, selecting colonies with antibacterial zones by plate diffusion method, and obtaining Escherichia coli F10 with antibacterial activity through separation and identification.
[0009] According to the above construction method, preferably, the Fosmid library is a human intestinal microbial Fosmid library. More preferably, the microbial sample source for constructing the human intestinal microbial Fosmid library is a healthy human fecal sample, and the healthy human fecal sample is used according to Copycontrol TM The Fosmid Library Production Kit (Epicentre) was used to construct the Fosmid library.
[0010] According to the above construction method, preferably, the construction method of the Fosmid library is: extracting genomic DNA from healthy human feces samples; breaking the genomic DNA into fragments to obtain DNA fragments; performing electrophoresis separation on the DNA fragments after end repair, and recovering DNA fragments of a specific size; connecting the recovered DNA fragments of a specific size to a plasmid, packaging, screening positive clones and storing them to obtain a Fosmid library.
[0011] The third aspect of the present invention provides the use of the recombinant engineered Escherichia coli described in the first aspect in the synthesis of indole compounds.
[0012] According to the above application, preferably, the indole compound is indole-3-carbaldehyde or / and indole trimer (trisindoline),
[0013] The structural formula of the indole trimer is as follows:
[0014]
[0015] The structural formula of the indole-3-carboxaldehyde is as follows:
[0016]
[0017] The fourth aspect of the present invention provides a method for preparing indole compounds, which comprises: inoculating the recombinant engineered Escherichia coli described in the first aspect into a fermentation medium for fermentation culture to obtain a fermentation broth, collecting the supernatant after centrifugation of the fermentation broth, adding ethyl acetate to the supernatant for extraction, collecting the supernatant after the extraction, concentrating the supernatant to obtain an extract; subjecting the extract to silica gel column chromatography separation, collecting the elution fraction of the silica gel column chromatography; subjecting the elution fraction to gel column chromatography and high performance liquid chromatography separation and purification to obtain an indole compound.
[0018] According to the above preparation method, preferably, the chromatography column used in the silica gel column chromatography is a normal silica gel column, and the eluent used in the silica gel column chromatography is a mixture of dichloromethane and methanol. More preferably, when the indole compound is indole-3-carboxaldehyde, the eluent used in the silica gel column chromatography is a mixture of dichloromethane and methanol in a volume ratio of 80:1; when the indole compound is an indole trimer, the eluent used in the silica gel column chromatography is a mixture of dichloromethane and methanol in a volume ratio of 40:1.
[0019] According to the above preparation method, preferably, the chromatography column used in the gel column chromatography is a Sephadex LH-20 gel column, and the eluent used in the gel column chromatography is a mixture of dichloromethane and methanol. More preferably, the eluent used in the gel column chromatography is a mixture of dichloromethane and methanol in a volume ratio of 2:1.
[0020] According to the above preparation method, preferably, the high performance liquid chromatography is semi-preparative high performance liquid chromatography; when the indole compound is indole-3-carboxaldehyde, the chromatographic column used for semi-preparative high performance liquid chromatography detection is a YMC-Pack ODS column, and the mobile phase is a methanol aqueous solution with a volume fraction of 55%; when the indole compound is an indole trimer, the chromatographic column used for semi-preparative high performance liquid chromatography detection is a YMC-Pack ODS column, and the mobile phase is an acetonitrile aqueous solution with a volume fraction of 50%.
[0021] Compared with the prior art, the present invention has the following positive and beneficial effects:
[0022] (1) The present invention provides a new recombinant engineered Escherichia coli strain, namely, Escherichia coli F10, which can produce indole-3-carboxaldehyde and indole trimer metabolites during fermentation and can be used to prepare indole-3-carboxaldehyde and indole trimer.
[0023] (2) The preparation method of the indole compound of the present invention is to use the recombinant engineered Escherichia coli, namely Escherichia coli F10, as the fermentation strain for fermentation culture, and separate and purify the fermentation product to obtain the indole compound. Compared with the existing preparation method by chemical process, the indole compound is green and environmentally friendly, has the advantages of simple reaction conditions, few by-products, and high product purity, and provides a new preparation method for indole-3-carboxaldehyde and indole trimer. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 is the HPLC chromatogram of the elution fraction L4;
[0025] Figure 2 This is the high-resolution mass spectrum of component A;
[0026] Figure 3 is the H NMR spectrum of component A;
[0027] Figure 4 is the NMR carbon spectrum of component A;
[0028] Figure 5 is the HPLC chromatogram of the elution fraction L5;
[0029] Figure 6 This is the high-resolution mass spectrum of component B;
[0030] Figure 7 is the H NMR spectrum of component B;
[0031] Figure 8 This is the NMR carbon spectrum of component B. DETAILED DESCRIPTION
[0032] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.
[0033] The following detailed description is exemplary and is intended to provide further description of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention.
[0034] The experimental methods in the following examples without specifying specific conditions all adopt conventional techniques in the art or follow the conditions recommended by the manufacturers; the reagents or instruments used without specifying the manufacturers are all conventional products that can be obtained commercially.
[0035] Example 1: Fosmid library construction
[0036] 1. Sources of intestinal microorganisms for construction of Fosmid library and extraction of intestinal microbial genomic DNA:
[0037] Fecal samples from three healthy people were selected, and the three fecal samples were subjected to differential centrifugation to wash and enrich microbial cells; the enriched microbial cells were broken by combining liquid nitrogen repeated freeze-thaw lysis, glass bead grinding and lysis solution high temperature lysis to obtain broken cells; the total genomic DNA of the broken cells was first extracted, and then the total genomic DNA was purified to extract microbial metagenomic DNA from intestinal contents. Three types of intestinal microbial metagenomic DNA were extracted from the three fecal samples and used to construct Fosmid libraries.
[0038] 2. Fosmid library construction
[0039] The microbial metagenomic DNA extracted above was used according to Copycontrol TM The Fosmid Library Production Kit (Epicentre) instructions are as follows:
[0040] (1) Metagenomic DNA shearing:
[0041] The extracted intestinal microbial metagenomic DNA was dissolved in TE solution at a concentration of 0.5 g / L, and was broken into about 35 kb by ultrasound and detected by pulsed field electrophoresis.
[0042] (2) DNA end repair:
[0043] The DNA fragment sample obtained by shearing in step (1) was end-filled using an End-repair enzyme Mix kit (New England Biolabs LTD., E6050L). The DNA end-filling reaction system is shown in Table 1 below.
[0044] Table 1 DNA end filling reaction system
[0045] volume sample 2μL Sterile water 8μL 10XBuffer 8μL dNTPMix 8μL ATP 50μL Insert DNA 4μL End-repair enzyme Mix
[0046] After incubation at room temperature for 45 minutes, add electrophoresis buffer and incubate at 70°C for 10 minutes to inactivate the End-repair enzyme Mix.
[0047] (3) Selection of end-repaired DNA fragments:
[0048] The end-repaired DNA of the desired size was separated by low melting point agarose gel electrophoresis. First, a 1% LMP agarose gel was prepared and placed in a 1×TAE solution. Secondly, 100 ng of Fosmid Control DNA (Epicentre,) was added to the gel wells on both sides and the end-repaired insert DNA was added in between. Finally, the samples were separated by gel electrophoresis at a stable voltage of 30-35 V, and a 2-4 mm gel slice was cut from the 36 Kb position and slightly above for recovery.
[0049] (4) Recovery of “separated size DNA”:
[0050] Place the gel cut from step (3) in a centrifuge tube and incubate at 70°C for 15 minutes to fully dissolve it; quickly transfer the tube to a 45°C water bath and add an appropriate volume of 1× GElase Buffer; add 1 μL of GElase Enzyme to every 100 μL of melted agarose and incubate with gentle stirring for 1 hour. Place the reaction system in a 70°C water bath to inactivate the GElase Enzyme. Pipette 500 μL of the solution into a sterile 1.5 ml tube, cool the tube in an ice bath for 5 minutes, and then place the tube in a centrifuge tube and centrifuge at maximum speed for 20 minutes to aggregate any insoluble low-grade polysaccharides into small spheres. These small spheres may be viscous, translucent to opaque. Carefully transfer 90%-95% of the upper layer of liquid floating on the surface to a sterile 1.5 ml tube, which contains the desired DNA. Determine the DNA concentration by fluorescence.
[0051] (5) Ligation reaction:
[0052] The recovered 36 kb DNA fragment was ligated with the plasmid pCC1FOS. The ligation reaction system is shown in Table 2 below:
[0053] Table 2 Ligation reaction system
[0054] volume sample 1μL Sterile water 1μL Fast-Link10×ligationBuffer 1μL ATP 1μL pCC1FOScloning-readyVector 5μL Insert DNA 1μL Fast-Link DNA Ligase
[0055] After reacting at room temperature for 2 hours, the reaction system was transferred to 70°C for 10 minutes to inactivate the Fast-Link DNA Ligase enzyme and obtain a ligation reaction solution.
[0056] (6) Construction of Copycontrol Fosmid Library:
[0057] One day before the packaging reaction, the host strain EPI300-T1R PlatingStrain cultured overnight in the preparation step was inoculated into 50 ml of LB culture medium containing 10 mmol / L magnesium sulfate and cultured at 37°C in a shaking incubator until the OD 600 Between 0.8-1.0, store at 4℃, and can be stored for up to 72 hours. Thaw 1 tube of Maxplax Lambda packaging extracts on ice for ligation reaction. During the thawing process, immediately transfer 25 microliters (half) of the phage extract packaging extracts to another 1.5 ml tube, and return the remaining 25 microliters of packaging extracts to the -70℃ refrigerator. Add 10 microliters of the ligation reaction solution obtained in step (5) to the 25 microliters of packaging extracts that have been melted on ice, and mix the liquid several times with a pipette, taking care to avoid bubbles. Package at 30℃ for 90 minutes. Add the remaining 25 microliters of packaging extracts to the tube and package at 30℃ for 90 minutes. Add phage dilution buffer (prepared with Phage Dilution Buffer) to 1 ml and mix well. Add 25 microliters of chloroform to each tube, mix well and store at 4℃.
[0058] (7) Titer determination of successfully packaged Copycontrol Fosmid clones:
[0059] A series of dilutions were obtained by adding the successfully packaged phage particles to a sterile tube containing phage dilution solution. 10 μl of the above dilution solution was added to the 100 μl host bacteria solution prepared in step (6) and cultured at 37°C for 20 minutes. The host bacteria EPI300-T1R infected with phage was spread on an LB plate containing 12.5 μg / ml chloramphenicol, cultured at 37°C overnight, and the Copycontrol Fosmid clone was picked, the number of clones was counted, and the concentration of successfully packaged phage particles was calculated.
[0060] (8)Copycontrol Fosmid Library Coating and Screening:
[0061] The phage particles obtained in (7) were diluted with phage diluent PDB to obtain the required number of clones and the required density of clones on the plate. The two were mixed at a ratio of 10 μl of diluted phage particles per 100 μl of EPI300-T1R host bacteria solution. The phages were adsorbed at 37°C for 20 minutes. The bacteria infected with phages were spread on LB plates containing 12.5 μg / ml chloramphenicol and cultured at 37°C overnight to select Copycontrol Fosmid clones.
[0062] The present invention isolates intestinal microorganisms from stool samples of three healthy young people, extracts their total genomic DNA, and constructs three Fosmid libraries with capacities of 1,228,000, 636,000 and 748,000, respectively. The three Fosmid libraries are stored in a 96-well plate in the form of 2,000 clones / well, 1,000 clones / well and 1,000 clones / well, respectively.
[0063] Example 2: Construction and screening of recombinant Escherichia coli
[0064] 200 positive clones were selected from each of the three Fosmid libraries and introduced into the BL21 host bacteria to obtain 600 recombinant strains. After culturing various indicator bacteria (Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus mycoides) in LB medium, 200 μL was taken and cultured until OD 600The indicator bacteria suspension of 0.6-1.0 was applied on LB solid medium. After the sterile circular filter paper (6mm in diameter) was soaked in LB liquid medium, it was placed on LB solid medium coated with indicator bacteria as a blank control. The single colony introduced into BL21 was inoculated into LB medium, and cultured in a shaking table at 180r / min until OD600 reached 0.6-1.0. The fermentation liquid was soaked with filter paper and placed on LB medium coated with indicator bacteria. The colonies with transparent antibacterial circles around them were screened, which were strains with antibacterial activity. A total of 170 heterologous clones that affected the growth of indicator bacteria were screened using the above method. Among the 170 screened heterologous clones, a colony was found to be a light yellow strain F10. The light yellow strain F10 was picked and preserved and used for fermentation culture. Through subsequent fermentation culture and separation and identification of fermentation products, it was found that the strain F10 was able to synthesize indole-3-carboxaldehyde and indole trimer during the fermentation process. The inventor named the strain F10 Escherichia coli F10 and preserved it in the China Center for Type Culture Collection with the preservation number CCTCC NO: M2024964 and the preservation date of May 16, 2024.
[0065] Example 3: Fermentation of Escherichia coli F10, separation and identification of fermentation products
[0066] 1. Fermentation culture of Escherichia coli F10:
[0067] Escherichia coli F10 was inoculated into LB medium, and cultured in a shaker at 220 rpm / min and 37° C. for 48 hours, and then the fermentation liquid was collected; the LB medium contained 5 g of yeast extract, 10 g of tryptone, and 10 g of sodium chloride per liter, and the pH was 7.0.
[0068] 2. Separation and identification of fermentation products:
[0069] (1) The bacterial liquid and bacterial cells of the fermentation broth were separated by a centrifuge (8000 rpm, 5 min), and the bacterial liquid was ultrasonically extracted with an equal volume of ethyl acetate for 4-5 times until the extract was basically colorless. The extract was concentrated by a rotary evaporator to obtain a total extract.
[0070] (2) The total extract obtained in step (1) is subjected to normal silica gel column chromatography (the filler of the normal silica gel column is silica gel with a particle size of 200-300 mesh, the brand of silica gel is Greagent, and the CAS number of silica gel is 63231-67-4), and a dichloromethane-methanol system is used as an eluent for isocratic elution. During the isocratic elution, the volume ratio of dichloromethane to methanol in the dichloromethane-methanol system is set as follows: 100% CH2Cl2, 100:1, 90:1, 80:1, 60:1, 40:1, 20:1, 15:1, 4:1, 100% MeOH; the elution fraction with an elution gradient of 80:1 in the elution process is collected, and the fraction is concentrated by rotary evaporation to obtain component S4; the elution fraction with an elution gradient of 40:1 in the elution process is collected, and the fraction is concentrated by rotary evaporation to obtain component S6;
[0071] The S4 component was loaded onto a Sephadex LH-20 gel column for chromatographic purification. During the chromatographic purification, an eluent (the eluent was prepared by mixing dichloromethane and methanol in a volume ratio of 2:1) was used for elution. The elution flow rate was 1 min / mL, and 120 mL of each fraction was collected to obtain 5 elution fractions, namely, elution fraction L1, elution fraction L2, elution fraction L3, elution fraction L4, and elution fraction L5. The elution fraction L4 was purified by a semi-preparative high performance liquid chromatograph on a YMC-Pack ODS column at a flow rate of 2.5 mL / min and a mobile phase of a methanol-water solution with a volume fraction of 55%. The liquid chromatogram is shown in FIG. Figure 1 As shown, the component with a retention time of 14.144 min was collected and recorded as component A. High-resolution mass spectrometry and nuclear magnetic resonance spectrum detection were performed on component A. The mass spectrometry detection results are shown in Figure 2 As shown by Figure 2 It can be seen that the molecular weight of the compound corresponding to component A is ESI-MS m / z: 144.0477 [MH] - The results of H NMR and C NMR of component A are as follows: Figure 3 Figure 4 As shown, the NMR data are assigned as follows: 1 H-NMR (400MHz, CD3OD)δ H :9.87(s,1H),8.25–7.89(m,2H),7.50–7.37(m,1H),7.35–7.03(m,2H),3.34–3.23(m,1H); 13 C-NMR (101MHz, CD3OD)δ C:187.42,139.76,138.91,125.67,124.99,123.61,122.37,120.07,113.13. The mass spectrometry and NMR detection results of component A are consistent with the reported indole-3-carbaldehyde data. Therefore, it is determined that the substance corresponding to component A is indole-3-carbaldehyde.
[0072] The S6 component was loaded onto a Sephadex LH-20 gel column for chromatographic purification. During the chromatographic purification, an eluent (the eluent was prepared by mixing dichloromethane and methanol in a volume ratio of 2:1) was used for elution. The elution flow rate was 1 min / mL, and 100 mL of each fraction was collected to obtain 6 elution fractions, namely, elution fraction L1, elution fraction L2, elution fraction L3, elution fraction L4, elution fraction L5, and elution fraction L6. The elution fraction L5 was purified using a semi-preparative high performance liquid chromatograph on a YMC-PackODS column at a flow rate of 3 mL / min and a mobile phase of 50% by volume acetonitrile-water solution. The liquid chromatogram is shown in FIG. Figure 5 As shown, the component with a retention time of 16.130 minutes was collected and recorded as component B, and high-resolution mass spectrometry and nuclear magnetic resonance spectrum detection were performed on component B. The high-resolution mass spectrometry detection results are shown in Figure 6 As shown by Figure 6 It can be seen that the molecular weight of the compound corresponding to component B is ESI-MS m / z: 386.1209 [M+Na] + The H NMR spectrum data of component B is as follows Figure 7 As shown, the NMR carbon spectrum data is as follows Figure 8 As shown, the NMR data are assigned as follows: 1 H-NMR (400MHz, CD3OD)δ H :7.30(d,J=8.2Hz,2H),7.27–7.23(m,2H),7.23–7.20(m,1H),7.02(dd,J=4.3,3.2Hz,1H),6.99(d d,J=8.1,1.0Hz,1H),6.94(td,J=7.6,1.0Hz,1H),6.87(s,2H),6.78(ddd,J=8.0,7.1,0.9Hz,2H); 13 C-NMR (101MHz, CD3OD)δ C182.73,142.27,138.75,136.72,129.00,127.26,126.45,125.58,123.3,122.35,122.07,119.53,115.61,112.34,110.98,54.93. The mass spectrometry and NMR detection results of component B were consistent with the reported indole trimer data, and the compound corresponding to component B was determined to be indole trimer (trisindoline).
[0073] In summary, the present invention effectively overcomes the deficiencies in the prior art and has a high industrial utilization value. The above embodiments serve to illustrate the substantive content of the present invention, but do not limit the protection scope of the present invention. Those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and protection scope of the technical solution of the present invention.
Claims
1. A recombinant Escherichia coli bacterium, characterized in that: The recombinant engineered Escherichia coli is Escherichia coli F10, which is stored in China Center for Type Culture Collection with a storage number of CCTCC NO: M2024964.
2. Use of the recombinant engineered Escherichia coli bacteria according to claim 1 in the preparation of indole compounds.
3. The use according to claim 2, characterized in that: The indole compound is indole-3-carboxaldehyde and / or indole trimer, and the structural formula of the indole trimer is as follows:
4. A method for preparing an indole compound, characterized in that: The recombinant engineered Escherichia coli bacteria of claim 1 are inoculated into a fermentation medium for fermentation culture to obtain a fermentation broth, the fermentation broth is centrifuged and the supernatant is collected, ethyl acetate is added to the supernatant for extraction, and after the extraction is completed, the supernatant is collected, and the supernatant is concentrated to obtain an extract; the extract is separated by silica gel column chromatography, and the eluted fraction of the silica gel column chromatography is collected; The eluted fraction is separated and purified by gel column chromatography and high performance liquid chromatography to obtain indole compounds.
5. The preparation method according to claim 4, characterized in that: The chromatography column used in the silica gel column chromatography is a normal silica gel column, and the eluent used in the silica gel column chromatography is a mixture of dichloromethane and methanol.
6. The preparation method according to claim 5, characterized in that: When the indole compound is indole-3-carboxaldehyde, the volume ratio of dichloromethane to methanol in the eluent used in the silica gel column chromatography is 80:1; when the indole compound is an indole trimer, the volume ratio of dichloromethane to methanol in the eluent used in the silica gel column chromatography is 40:
1.
7. The preparation method according to any one of claims 4 to 6, characterized in that: The chromatography column used in the gel column chromatography is a Sephadex LH-20 gel column, and the eluent used in the gel column chromatography is a mixture of dichloromethane and methanol.
8. The preparation method according to claim 7, characterized in that: The volume ratio of dichloromethane to methanol in the eluent used in the gel column chromatography is 2:
1.
9. The preparation method according to claim 8, characterized in that: The high performance liquid chromatography is a semi-preparative high performance liquid chromatography; when the indole compound is indole-3-carboxaldehyde, the chromatographic column used in the semi-preparative high performance liquid chromatography detection is a YMC-Pack ODS column, and the mobile phase is a methanol aqueous solution with a volume fraction of 55%; When the indole compound is an indole trimer, the chromatographic column used in the semi-preparative high performance liquid chromatography detection is a YMC-Pack ODS column, and the mobile phase is an acetonitrile aqueous solution with a volume fraction of 50%.