Indole alkaloid compound, preparation method and application thereof

Abstract

The present invention discloses an indole alkaloid compound, a preparation method and an application thereof. It belongs to the field of microbial pesticide technology. The molecular structure of the indole alkaloid compound, the preparation of related microorganisms and the preparation method are provided. The indole alkaloid compound to which the present invention relates is different from the known indole alkaloid compounds in terms of structural features such as its structural skeleton and cyclization mode. The indole alkaloid compound involved is produced by fermentation of the strain Aspergillus japonicus TE‑739D and is easy to carry out large-scale fermentation production; the preparation method provided by the present invention can quickly and accurately prepare the indole alkaloid compound. It is found for the first time that the indole alkaloid compound has antibacterial activity against drug-resistant gray mold fungi, can significantly inhibit the growth of drug-resistant gray mold fungi, and can be used as a lead compound or a new biopesticide component with the ability to inhibit drug-resistant gray mold fungi.

Description

Technical Field

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

[0002] Gray mold, caused by the pathogen Botrytis cinerea, can harm over 200 commercial crops, including grapes, strawberries, and tomatoes, both during field growth and during fruit storage, causing significant economic losses. Consequently, gray mold is listed as one of the world's top ten plant fungal diseases, causing over $10 billion in economic losses worldwide annually. Currently, field control of gray mold relies primarily on synthetic pesticides such as azoxystrobin and boscalid. While these pesticides offer effective control of gray mold, their relatively limited mechanisms of action and the high genetic variability of Botrytis cinerea have led to the continuous development of resistance strains, resulting in multidrug-resistant strains that increase the difficulty of control. Currently, gray mold has developed multidrug resistance to a variety of fungicides, including boscalid, azoxystrobin, iprodione, and pyrimethanil. Therefore, the search for new potential agricultural antibiotics has become an important approach to gray mold control. Furthermore, the excessive use of synthetic pesticides poses significant challenges, including pesticide residues and environmental pollution.

[0003] Searching for active secondary metabolites from microorganisms that can inhibit plant pathogens has become an important source for the development of new biopesticides. Microbial secondary metabolites have the advantages of being easy to ferment and produce, having low toxicity, being easy to degrade, and having 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, with the collection 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 alkaloids 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 urgently need to solve. Summary of the Invention

[0006] In light of this, the present invention provides an indole alkaloid compound, its preparation method, and its use. The indole alkaloid compound is a novel compound that has not been reported previously. The present invention provides a preparation method for the compound and, for the first time, demonstrates its inhibitory activity against drug-resistant Botrytis cinerea. The compound has the potential to be developed into a new natural product biopesticide, providing a new compound entity for the biological control of plant fungal diseases.

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

[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 mold Aspergillus japonicus TE-739D for preparing the above-mentioned indole alkaloid compounds; the preservation number of the Japanese mold Aspergillus japonicus TE-739D is CGMCC: NO.40901.

[0011] The present invention also provides a method for preparing indole alkaloid compounds using the above-mentioned 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: first eluting with dichloromethane-methanol and then with petroleum ether-ethyl acetate in a gradient manner, and collecting the petroleum ether-ethyl acetate elution fraction;

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

[0016] (5) Collecting the eluted fractions of methanol-water reverse phase silica gel and performing preparative thin layer chromatography purification to obtain an indole alkaloid compound having 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, 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 vacuum concentration condition is: vacuum degree -0.1 MPa, and concentrating to a non-fluid state.

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

[0020] Preferably: step (4) is specifically as follows: the elution component with a volume ratio of petroleum ether to ethyl acetate of 1:1 is subjected to reverse-phase silica gel column chromatography, and gradient elution is performed 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, and 1:0, and the amount of mixed solvent used in each gradient is 1.2 L.

[0021] Preferably, step (5) is specifically as follows: collecting the reverse phase silica gel elution fraction 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 is 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, which achieves the following technical effects:

[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 cyclization 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 present invention can quickly and accurately prepare the indole alkaloid compounds.

[0027] For the first time, indole alkaloids have been found to exhibit antifungal activity against drug-resistant Botrytis cinerea, significantly inhibiting its growth with a MIC of 16 μg / mL. This activity is superior to the commonly used chemical fungicides azoxystrobin (MIC = 64 μg / mL) and boscalid (MIC = 32 μg / mL). These compounds could serve as lead compounds or new biopesticide ingredients for inhibiting drug-resistant Botrytis cinerea. 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 following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any 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 figure is a hydrogen spectrum of the indole alkaloid compound aspertaichamide B provided by the present invention ( 1 HNMR) diagram.

[0032] Figure 4 The accompanying 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 clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. 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 making creative efforts are within the scope of protection of the present invention.

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

[0035] Example 1

[0036] 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. The culture was then placed in a static fermentation culture at 28°C for 30 days.

[0038] The liquid culture medium is composed of 4% glucose (mass percentage), 1% peptone (mass percentage), 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-fluid state) to obtain a non-fluid fermentation crude extract.

[0040] (3) The crude fermentation extract was separated by silica gel column chromatography (80 mm inner diameter, 600 mm length, glass chromatography column with a sand plate and a nozzle): in the order of increasing polarity of the eluent, first with dichloromethane-methanol in a volume ratio 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 a volume ratio 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 of 1:1 was subjected to reverse phase silica gel column chromatography (30 mm inner diameter, 600 mm length, glass chromatography column with a standard PTFE valve), and gradient elution was performed using a methanol-water 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, for a total of 10 elution fractions, and the amount of mixed solvent used for each gradient was 1.2 L).

[0042] (5) The fractions eluted from reversed-phase silica gel 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 (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 ), suggesting 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: 125 MHz; solvent used for NMR testing: deuterated methanol)

[0046]

[0047] Example 2

[0048] The inhibitory activity of the indole alkaloids against drug-resistant Botrytis cinerea was determined by the minimum inhibitory concentration (MIC) method, ie, 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 Province. It is resistant to the commonly used chemical synthetic fungicides for gray mold, including boscalid, myclobutanil, iprodione, and pyrimethanil.

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

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

[0052] The compound and a positive control were added to a suspension of drug-resistant Botrytis cinerea at varying concentrations. After incubation, the compound was observed. If the drug-resistant Botrytis cinerea grew in a particular well, the drug concentration in that well was ineffective in inhibiting its growth. The liquid in that well became turbid, and the transmittance decreased significantly. Conversely, the liquid in that well became clear, and the transmittance decreased insignificantly. The lowest sample concentration that completely inhibited the growth of drug-resistant Botrytis cinerea in that well was the compound's MIC.

[0053] MIC testing results showed that aspertaichamide B, an indole alkaloid compound, exhibited inhibitory activity against drug-resistant Botrytis cinerea, significantly inhibiting its growth with an MIC of 16 μg / mL. This activity was superior to the commonly used chemical fungicides azoxystrobin (MIC = 64 μg / mL) and boscalid (MIC = 32 μg / mL). This compound could serve as a lead compound or new biopesticide ingredient for inhibiting drug-resistant Botrytis cinerea.

[0054] In summary, the present invention can quickly separate and prepare the above-mentioned indole alkaloid compounds by fermenting and culturing the above-mentioned strain TE-739D, extracting and purifying the (liquid) fermentation product, and can inhibit the activity of gray mold-resistant bacteria.

[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 is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily 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 is not limited to the embodiments shown herein but is intended to conform 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: Formula I: .

2. A Japanese Aspergillus Aspergillus japonicus TE-739D is characterized by: For preparing the indole alkaloid compound according to claim 1; the Aspergillus japonicus Aspergillus japonicus The deposit number of TE-739D is CGMCC: NO.40901.

3. A method of using the Aspergillus japonicus according to claim 2 Aspergillus japonicus TE-739D The method for preparing indole alkaloid compounds is characterized in that: The following steps are involved: (1) Aspergillus japonicus Aspergillus japonicus TE-739D was fermented and cultured to obtain 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: first using dichloromethane-methanol and then petroleum ether-ethyl acetate for gradient elution, and collecting the petroleum ether-ethyl acetate elution fraction; (4) The eluted fraction of petroleum ether-ethyl acetate was subjected to reverse phase silica gel column chromatography, and gradient elution was performed with methanol-water; (5) Collecting the eluted fractions of methanol-water reverse phase silica gel and performing preparative thin layer chromatography to purify the product to obtain the indole alkaloid compound having a molecular structure as shown in Formula I as claimed in claim 1.

4. The method according to claim 3, wherein Step (1) is specifically as follows: take Aspergillus japonicus Aspergillus japonicus TE-739D was inoculated into a liquid culture medium for fermentation culture. The liquid culture medium contained 4% by weight of glucose, 1% by weight of peptone, and the remainder was distilled water.

5. The method according to claim 4, wherein 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. The vacuum decompression concentration conditions are: vacuum degree -0.1 MPa, and concentrating to a non-fluid state.

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

1.

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

8. The method according to claim 7, wherein Step (5) is specifically as follows: collecting the reverse phase silica gel elution fraction 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, developing system is dichloromethane-methanol volume ratio 20:1, 40 mL.

9. Use of the indole alkaloid compound according to claim 1 for inhibiting drug-resistant gray mold.

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