Indole alkaloid compound as well as preparation method and application thereof

Abstract

The invention discloses an indole alkaloid compound as well as a preparation method and application thereof. Belongs to the technical field of microbial pesticides. The invention provides a molecular structure of an indole alkaloid compound, a preparation related microorganism and a preparation method of the indole alkaloid compound. According to the indole alkaloid compound provided by the invention, the structural characteristics such as a structural framework and a ring forming mode are different from those of known indole alkaloid compounds. The related indole alkaloid compound is produced by fermenting a strain Aspergillus japonicus TE-739D, and is easy to carry out large-scale fermentation production; the preparation method provided by the invention can be used for rapidly and accurately preparing the indole alkaloid compound. It is found for the first time that the indole alkaloid compound has bacteriostatic activity on drug-resistant botrytis cinerea, can significantly inhibit the growth of the drug-resistant botrytis cinerea, and can be used as a lead compound or a novel biopesticide component for inhibiting the drug-resistant botrytis cinerea.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial pesticides, and more particularly to an indole alkaloid compound and a preparation method and application thereof. Background Art

[0002] The pathogen of gray mold is Botrytis cinerea, which can harm the field growth and fruit storage of more than 200 economic crops such as grapes, strawberries, tomatoes, etc., and cause serious economic losses. Therefore, gray mold is listed as one of the world's top ten plant fungal diseases, and can cause more than 10 billion US dollars in economic losses worldwide each year. At present, field control of gray mold is still mainly based on chemical synthetic pesticides such as azoxystrobin and boscalid. Although chemical synthetic pesticides can effectively control gray mold, due to their relatively simple mechanism of action and the strong genetic variability of Botrytis cinerea, strain resistance continues to develop, and the resulting multi-drug resistant bacteria increase the difficulty of control. Up to now, gray mold has developed multi-drug resistance to a variety of fungicides such as boscalid, azoxystrobin, isoprodinil and pyrimethanil, so finding new potential molecules for agricultural antibiotics has become an important way to prevent and control gray mold. At the same time, the problems of pesticide residues and environmental pollution caused by excessive use of chemical synthetic pesticides should not be ignored.

[0003] Searching for active secondary metabolites from microorganisms that inhibit plant pathogens has become an important source for the development of new biopesticides. Microbial secondary metabolites have the advantages of easy fermentation production, low toxicity, easy degradation, and high environmental compatibility; at the same time, microbial secondary metabolites often have a "multi-point attack" mechanism of action and have become one of the effective means of preventing and controlling drug-resistant bacteria. Chinese invention patent CN114369051B discloses a pyrrolidone compound obtained from the liquid fermentation product of strain Aspergillus sp. TR15 (deposited in the China Center for Type Culture Collection, deposit number: CCTCCNO: M20211402), a preparation method, and its activity in inhibiting the mycelial growth and spore germination of drug-resistant gray mold fungi.

[0004] It is reported that indole alkaloid compounds have multiple biological activities such as insecticide, anti-tumor and antibacterial.

[0005] Therefore, whether it is possible to provide an indole alkaloid compound and use it to solve the above problems is an issue that those skilled in the art need to solve urgently. Summary of the invention

[0006] In view of this, the present invention provides an indole alkaloid compound and a preparation method and application thereof. The indole alkaloid compound involved is a new compound that has not been reported. The present invention provides a preparation method of the compound and discovers for the first time its inhibitory activity against drug-resistant gray mold fungi. The compound has the potential to be developed into a new natural product biological pesticide. A new compound entity is provided for the biological control of plant fungal diseases.

[0007] Deposit information: Aspergillus japonicus TE-739D is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC: NO.40901 and the deposit date October 25, 2023. In order to achieve the above purpose, the present invention adopts the following technical scheme:

[0008] An indole alkaloid compound, whose molecular structure is as shown in Formula I:

[0009] Formula I:

[0010] The present invention also provides a Japanese aspergillus TE-739D for preparing the above-mentioned indole alkaloid compounds; the preservation number of the Japanese aspergillus TE-739D is CGMCC: NO.40901.

[0011] The present invention also provides a method for preparing indole alkaloid compounds by using the Aspergillus japonicus TE-739D, comprising the following steps:

[0012] (1) fermenting Aspergillus japonicus TE-739D to obtain a liquid fermentation product;

[0013] (2) extracting the liquid fermentation product with ethyl acetate and concentrating it under vacuum to obtain a non-flowing fermentation crude extract;

[0014] (3) separating the components of the crude fermentation extract by silica gel column chromatography: firstly eluting with dichloromethane-methanol and then with petroleum ether-ethyl acetate by gradient elution, and collecting the elution fraction of petroleum ether-ethyl acetate;

[0015] (4) subjecting the eluted fraction of petroleum ether-ethyl acetate to reverse phase silica gel column chromatography, and performing gradient elution with methanol-water;

[0016] (5) Collect the eluted fractions of methanol-water reverse phase silica gel and purify them by preparative thin layer chromatography to obtain an indole alkaloid compound with a molecular structure as shown in Formula I.

[0017] Preferably, step (1) is specifically as follows: Aspergillus japonicus TE-739D is inoculated into a liquid culture medium for fermentation culture, wherein the liquid culture medium comprises 4% by weight of glucose, 1% by weight of peptone, and the remainder is distilled water.

[0018] Preferably, step (2) is specifically as follows: extracting the liquid culture with ethyl acetate three times, 300 mL each time, combining the extracts and concentrating under vacuum pressure, wherein the conditions for vacuum concentration are: vacuum degree -0.1 MPa, and concentrating to a non-fluid state.

[0019] Preferably, step (3) is specifically as follows: firstly, dichloromethane-methanol with a volume ratio of 20:1, 10:1, 5:1, 2:1 and 1:1, each gradient of 1 L is used, and then, petroleum ether-ethyl acetate with a volume ratio of 30:1, 10:1, 5:1, 2:1 and 1:1, each gradient of 1 L is used for gradient elution, and the elution fraction with petroleum ether-ethyl acetate = 1:1 is collected.

[0020] Preferably, step (4) is specifically as follows: subjecting the elution component having a petroleum ether-ethyl acetate volume ratio of 1:1 to reverse phase silica gel column chromatography, and gradient eluting with alcohol-water having a volume ratio of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, 1:0, 10 elution components, and the amount of mixed solvent used for each gradient is 1.2 L.

[0021] Preferably, step (5) is specifically as follows: collecting the reverse phase silica gel elution component with a methanol-water volume ratio of 4:6, and performing preparative thin layer chromatography purification, thin layer chromatography: glass plate 20×20 cm, developing system dichloromethane-methanol volume ratio 20:1, 40 mL.

[0022] The present invention also provides the use of the above-mentioned indole alkaloid compound, or the above-mentioned Aspergillus japonicus TE-739D, or any of the above-mentioned methods in preparing a lead compound or biological pesticide for inhibiting pathogens.

[0023] Preferred: Pathogen: Drug-resistant Botrytis cinerea.

[0024] It can be seen from the above technical solutions that, compared with the prior art, the present invention discloses an indole alkaloid compound and a preparation method and application thereof, and the technical effects achieved are:

[0025] The indole alkaloid compounds involved are new compounds that have not been reported, as determined by searching the SciFindern database of natural products. Their structural features, such as the structural skeleton and ring-forming mode, are different from those of known indole alkaloid compounds.

[0026] The indole alkaloid compounds involved are produced by fermentation of the strain Aspergillus japonicus TE-739D, and are easy to carry out large-scale fermentation production; the preparation method provided by the invention can quickly and accurately prepare the indole alkaloid compounds.

[0027] For the first time, it was found that indole alkaloid compounds have antibacterial activity against drug-resistant gray mold, and can significantly inhibit the growth of drug-resistant gray mold, with a MIC value of 16μg / mL; the above activity is better than the commonly used chemical synthetic fungicides azoxystrobin (MIC=64μg / mL) and boscalid (MIC=32μg / mL). It can be used as a lead compound or new biopesticide ingredient that inhibits drug-resistant gray mold. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0029] Figure 1 The accompanying drawing is a molecular structure diagram of the indole alkaloid compound aspertaichamide B provided by the present invention.

[0030] Figure 2 The accompanying drawing is a high resolution mass spectrum (HRESIMS) diagram of the indole alkaloid compound aspertaichamide B provided by the present invention.

[0031] Figure 3 The accompanying drawing is a hydrogen spectrum of the indole alkaloid compound aspertaichamide B provided by the present invention ( 1 HNMR) graph.

[0032] Figure 4 The attached figure is a carbon spectrum of the indole alkaloid compound aspertaichamide B provided by the present invention ( 13 CNMR) diagram. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] The embodiment of the invention discloses an indole alkaloid compound and a preparation method and application thereof.

[0035] Example 1

[0036] The preparation process of an indole alkaloid compound (named aspertaichamide B):

[0037] (1) Under sterile conditions, a piece of Aspergillus japonicus TE-739D (about 1×1 cm) grown on a PDA plate was cut and inoculated into a conical flask containing liquid culture medium, and then fermented at 28°C for 30 days.

[0038] The liquid culture medium is 4% (mass percentage) of glucose, 1% (mass percentage) of peptone, and 300 mL of distilled water.

[0039] (2) The liquid culture medium (fermentation product) was extracted with ethyl acetate three times (300 mL each time), and the combined extracts were concentrated under vacuum (vacuum degree -0.1 MPa, concentrated to a non-flowing body) to obtain a non-flowing fermentation crude extract.

[0040] (3) The crude fermentation extract was separated by silica gel column chromatography (inner diameter 80 mm, length 600 mm, glass chromatography column with sand plate and nozzle): in the order of increasing polarity of the eluent, first with dichloromethane-methanol in volume ratios of 20:1, 10:1, 5:1, 2:1 and 1:1, each gradient of 1 L, and then with petroleum ether-ethyl acetate in volume ratios of 30:1, 10:1, 5:1, 2:1 and 1:1, each gradient of 1 L, and collect the elution fraction with petroleum ether-ethyl acetate = 1:1.

[0041] (4) The elution fraction with a ratio of petroleum ether to ethyl acetate = 1:1 was subjected to reverse phase silica gel column chromatography (inner diameter 30 mm, length 600 mm, glass chromatography column with a standard PTFE valve), and gradient elution was performed using methanol-water as a mixed solvent with a volume ratio of 1:9 to 1:0 (the volume ratios were 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, and 1:0, respectively, for a total of 10 elution fractions, and the amount of mixed solvent used for each gradient was 1.2 L).

[0042] (5) The reverse phase silica gel elution fractions with a methanol-water volume ratio of 4:6 were collected and purified by preparative thin layer chromatography (glass plate 20×20 cm, developing system: dichloromethane-methanol volume ratio 20:1, total 40 mL) to obtain compound aspertaichamide B (see Figure 1 ).

[0043] The molecular structure of the indole alkaloid compound aspertaichamide B was determined by high-resolution mass spectrometry HRESIMS and superconducting nuclear magnetic resonance spectroscopy NMR. The physicochemical properties of the indole alkaloid compound aspertaichamide B are as follows:

[0044] Properties: colorless oil; Solubility: easily soluble in methanol and acetone, slightly soluble in dichloromethane; Optical rotation: [α] 20 D +26.0 (methanol); UV absorption spectrum λ max (Methanol, logε) 230 (4.32), 260 (4.34), 388 (3.68) nm; High resolution mass spectrometry (HRESIMS): m / z 506.2289 [MH] - (Theoretical value C 28 H 32 N3O6,506.2291)(see Figure 2 ), indicating that its molecular formula is C 28 H 33 N3O6; H NMR ( 1 H NMR) (see Figure 3 ) data and carbon spectrum ( 13 C NMR) (see Figure 4 )Data are shown in Table 1.

[0045] Table 1: NMR data of indole alkaloid compound aspertaichamide B ( 1 H: 500MHz; 13 C: 125MHz; solvent used for NMR test: deuterated methanol)

[0046]

[0047] Example 2

[0048] The minimum inhibitory concentration (MIC) method was used to determine the inhibitory activity of the above-mentioned indole alkaloid compounds against drug-resistant Botrytis cinerea, that is, the lowest drug concentration that can inhibit the growth of pathogenic fungi in vitro.

[0049] The drug-resistant gray mold fungus WC1-4 was isolated from a grape greenhouse in Pingdu, Shandong. It is resistant to the commonly used chemical synthetic fungicides for gray mold, including boscalid, myclobutanil, cymethoxam, and pyrimethanil.

[0050] The drug-resistant gray mold fungus WC1-4 was inoculated into PDB medium and cultured at 28°C for 3 to 5 days. The bacterial solution was diluted with PDB medium to a concentration of 10 6CFU / mL, used for MIC determination. The above compound aspertaichamide B was dissolved in DMSO and prepared as a 2560μg / mL stock solution. A certain volume of the stock solution was taken and diluted with bacterial solution to different concentrations of 128μg / mL, 64μg / mL, 32μg / mL, 16μg / mL, 8μg / mL, 4μg / mL, 2μg / mL and 1μg / mL sample solutions.

[0051] Azoxystrobin and boscalid were used as positive controls and were also prepared at the same concentration.

[0052] By adding different concentrations of the compound and the positive control to the bacterial suspension of drug-resistant gray mold, and observing after cultivation, if the drug-resistant gray mold grows in a certain well, it means that the drug concentration in the well cannot inhibit the growth of the bacteria, the liquid in the well is turbid, and the transmittance decreases significantly. On the contrary, the liquid in the well is clear, and the transmittance decreases insignificantly. The lowest sample concentration that completely inhibits the growth of drug-resistant gray mold in the well is the MIC of the compound.

[0053] The MIC test results show that the above-mentioned indole alkaloid compound aspertaichamide B has inhibitory activity against drug-resistant gray mold fungi and can significantly inhibit the growth of drug-resistant gray mold fungi, with a MIC value of 16μg / mL. The above-mentioned activity is better than the commonly used chemical synthetic fungicides azoxystrobin (MIC=64μg / mL) and boscalid (MIC=32μg / mL). The above-mentioned compound can be used as a lead compound or a new biopesticide component that inhibits drug-resistant gray mold fungi.

[0054] In summary, the present invention can inhibit the activity of gray mold resistant bacteria by quickly separating and preparing the above-mentioned indole alkaloid compounds through fermentation and cultivation of the above-mentioned strain TE-739D, extraction and purification of the (liquid) fermentation product.

[0055] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0056] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An indole alkaloid compound, characterized in that: Its molecular structure is as shown in Formula I:

2. A Japanese Aspergillus TE-739D, characterized in that Used for preparing the indole alkaloid compound described in claim 1; the preservation number of the Aspergillus japonicus TE-739D is CGMCC: NO.40901.

3. A method for preparing indole alkaloid compounds using Aspergillus japonicus TE-739D as described in claim 2, characterized in that: The following steps are involved: (1) fermenting Aspergillus japonicus TE-739D to obtain a liquid fermentation product; (2) extracting the liquid fermentation product with ethyl acetate and concentrating it under vacuum to obtain a non-flowing fermentation crude extract; (3) separating the components of the crude fermentation extract by silica gel column chromatography: firstly eluting with dichloromethane-methanol and then with petroleum ether-ethyl acetate by gradient elution, and collecting the elution fraction of petroleum ether-ethyl acetate; (4) subjecting the eluted fraction of petroleum ether-ethyl acetate to reverse phase silica gel column chromatography, and performing gradient elution with methanol-water; (5) Collect the eluted fractions of methanol-water reverse phase silica gel and purify them by preparative thin layer chromatography to obtain an indole alkaloid compound with a molecular structure as shown in Formula I.

4. The method according to claim 3, characterized in that Step (1) specifically comprises: inoculating Aspergillus japonicus TE-739D into a liquid culture medium for fermentation culture, wherein the liquid culture medium comprises 4% by weight of glucose, 1% by weight of peptone, and the remainder is distilled water.

5. The method according to claim 4, characterized in that Step (2) is specifically as follows: extracting the liquid culture with ethyl acetate three times, 300 mL each time, combining the extracts and performing vacuum decompression concentration, wherein the vacuum degree of the vacuum decompression concentration is -0.1 MPa, and the liquid is concentrated to a non-fluid state.

6. The method according to claim 5, characterized in that Step (3) is specifically as follows: firstly, dichloromethane-methanol with a volume ratio of 20:1, 10:1, 5:1, 2:1 and 1:1, each gradient of 1 L, and then, petroleum ether-ethyl acetate with a volume ratio of 30:1, 10:1, 5:1, 2:1 and 1:1, each gradient of 1 L, are used for gradient elution, and the elution fraction with petroleum ether-ethyl acetate = 1:1 is collected.

7. The method according to claim 6, characterized in that Step (4) is specifically as follows: subjecting the elution component having a petroleum ether-ethyl acetate volume ratio of 1:1 to reverse phase silica gel column chromatography, and gradient eluting with alcohol-water having a volume ratio of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, 1:0, 10 elution components, and the amount of mixed solvent used for each gradient is 1.2 L.

8. The method according to claim 7, characterized in that Step (5) is specifically as follows: collecting the reverse phase silica gel elution component with a methanol-water volume ratio of 4:6, and performing preparative thin layer chromatography purification, wherein the thin layer chromatography: glass plate 20×20 cm, the developing system is dichloromethane-methanol volume ratio 20:1, 40 mL.

9. Use of the indole alkaloid compound of claim 1, or Aspergillus japonicus TE-739D of claim 2, or the method of any one of claims 3 to 8 in preparing a lead compound or biological pesticide for inhibiting pathogens.

10. The use according to claim 9, characterized in that The pathogen is drug-resistant Botrytis cinerea.

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